COLLAGEN PRODUCTION PROMOTING COMPOSITION COMPRISING Amidated AMINO ACID

Abstract

The present invention relates to a composition for promoting collagen production comprising an amidated amino acid, and more particularly, to a composition for promoting collagen production in various cells such as fibroblasts, hair follicle cells, dermal papilla cells, endothelial cells, chondrocytes, muscle cells, and osteoblasts, and more particularly, to a composition for promoting collagen production in various cells such as fibroblasts, hair follicle cells, dermal papilla cells, endothelial cells, chondrocytes, muscle cells, and osteoblasts. Therefore, various diseases and lesions caused by reduction of collagen in skin, hair, blood vessels, cartilage, tendons, connective tissues, muscles, bones, teeth and periodontal tissues are prevented, improved or treated.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0022559 filed on February 21, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a composition for promoting collagen production, comprising amidated amino acids, and more particularly to a composition for promoting collagen production in various cells such as fibroblasts, hair follicle cells, dermal papilla cells, endothelial cells, chondrocytes, myocytes and osteoblasts, and thereby preventing, improving or treating various diseases and lesions that may be caused by a decrease in collagen in skin, hair, blood vessels, cartilage, tendons, connective tissues, muscles, bones, teeth and periodontal tissues. Background of the Invention

[0004] Collagen is the most abundant protein in animals, accounting for 30% of the dry weight of the skin. Collagen exists in a triple helical structure, and its main amino acid sequence repeats as glycine-proline-X or glycine-X-hydroxyproline, where X can be any amino acid except glycine, proline or hydroxyproline. To date, a total of 28 different types of collagen have been identified, of which type I collagen is the most common, accounting for more than 90% of human collagen. Normal human skin contains various types of collagen, specifically 80-90% type I collagen, 8-12% type III collagen and 5% type V collagen. Collagen is the main component of the extracellular matrix (ECM) of the dermis, providing structural support and maintaining the firmness and elasticity of the skin.

[0005] Insufficient or excessive dermal collagen is associated with a variety of skin diseases and cosmetic problems. Collagen disorders occur when collagen production and maturation are defective due to genetic causes, when collagen production is insufficient due to a deficiency of ascorbic acid (AA), or when collagen production is excessively increased due to an autoimmune response.

[0006] Transforming growth factor-β1 (TGF-β1) increases cellular collagen production and promotes cell proliferation and differentiation. Ascorbic acid (vitamin C) plays a vital role in the post-translational stage of collagen as an essential cofactor for prolyl hydroxylases and lysyl hydroxylases. Ascorbic acid can also stimulate the transcription of collagen genes in cells.

[0007] Extracellular matrix proteins including collagen have a biased amino acid composition, so the production of collagen by cells is affected by the availability of specific amino acids. According to existing research reports, external supply of proline increases collagen production in fibroblasts, and glycine is known to have a greater effect on collagen production in articular chondrocytes than proline, lysine, and other amino acids.

[0008] However, existing methods including TGF-β1, ascorbic acid and free amino acids are not sufficient to promote collagen production, so there is a need to develop new substances that can more effectively stimulate intracellular collagen synthesis. Summary of the invention

[0009] Issues to be resolved

[0010] Therefore, the inventors of the present invention have discovered a substance that can safely and very effectively promote collagen production in various cells, and found that amidated amino acids can exhibit this effect.

[0011] Therefore, the object of the present invention is to provide a pharmaceutical composition, a cosmetic composition or a food composition for promoting collagen production, which comprises one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalaninamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionineamide, amidated asparagine (asparaginamide), prolineamide, amidated glutaminamide (glutaminamide), arginineamide, threonineamide, valineamide and tyrosinamide.

[0012] In addition, the present invention also aims to provide a pharmaceutical composition, a cosmetic composition or a food composition for promoting collagen production, which is composed of one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0013] In addition, the present invention aims to provide a pharmaceutical composition, a cosmetic composition or a food composition for promoting collagen production, which is essentially composed of one or more amidated amino acids selected from the group consisting of alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0014] The object of the present invention is to provide the use of one or more amidated amino acids selected from the following in the preparation of a composition for promoting collagen production: alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine, and tyrosinamide.

[0015] The object of the present invention is to provide a method for promoting collagen production, comprising administering to a subject in need thereof an effective amount of a promoting composition, wherein the promoting composition comprises one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0016] Means of solving the problem

[0017] To achieve the above-mentioned purpose of the present invention, the present invention provides a composition for promoting collagen production, which comprises one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0018] In addition, the present invention provides a composition for promoting collagen production, which is composed of one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0019] In addition, the present invention provides a composition for promoting collagen production, which is basically composed of one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0020] In order to achieve the above-mentioned object of the present invention, the present invention provides the use of one or more amidated amino acids selected from the following in the preparation of a composition for promoting collagen production: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0021] To achieve the above-mentioned purpose of the present invention, the present invention provides a method for promoting collagen production, which comprises administering to a subject in need thereof an effective amount of a promoting composition, wherein the promoting composition comprises one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0022] The present invention is described in detail below.

[0023] The present invention provides a pharmaceutical composition, a cosmetic composition or a food composition for promoting collagen production, which comprises one or more amidated amino acids selected from the group consisting of alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0024] The single-letter (three-letter) abbreviations of amino acids used in this specification are composed of the following amino acids according to the standard abbreviation rules in the field of biochemistry:

[0025] A (Ala): Alanine; C (Cys): Cysteine; D (Asp): Aspartic acid; E (Glu): Glutamic acid; F (Phe): Phenylalanine; G (Gly): Glycine; H (His): Histidine; I (Ile): Isoleucine; K (Lys): Lysine; L (Leu): Leucine; M (Met): Methionine; N (Asn): Asparagine; O (Ply): Pyrrolysine; P (Pro): Proline; Q (Gln): Glutamine; R (Arg): Arginine; S (Ser): Serine; T (Thr): Threonine; U (Sec): Selenocysteine; V (Val): Valine; W (Trp): Tryptophan; Y (Tyr): Tyrosine.

[0026] In addition, the amino acid sequences of all peptides in this specification are described from the amino terminus (N-terminus, amino terminus or N-terminus) to the carboxyl terminus (C-terminus, carboxyl terminus or C-terminus) in accordance with standard provisions in the field of biochemistry. If the carboxyl terminus of a peptide described in this specification is labeled with an amino group (-NH2), this does not refer to the amino terminus of the amino acid sequence, but refers to the addition of an amino group to the carboxyl terminus by amidation of the carboxyl terminus by a chemical synthesis method of the peptide.

[0027] According to one embodiment of the present invention, alaninamide, cysteine, glutamate-α-amide, phenylalaninamine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamine, amidated glutamine, arginine, threonine, valine and tyrosinamine promote collagen production in each of human fibroblasts, chondrocytes, myocytes, osteoblasts, hair follicle cells and endothelial cells.

[0028] Collagen is found in many parts of the body. More than 90% of the collagen in the body is type I. To date, 28 types of collagen have been identified and described. The five most common types of collagen are as follows:

[0029] Collagen I: skin, tendons, blood vessel connections, organs and bones (the main component of the organic part of bones);

[0030] Collagen II: cartilage (the main component of cartilage);

[0031] Collagen III: Usually found together with collagen I, it is the main component of reticular fibers;

[0032] Collagen IV: forms the basal lamina, which is the epithelial secretory layer of the basement membrane; and

[0033] Collagen V: cell surface, hair and placenta

[0034] Collagen is essential to health as it plays a key role in maintaining the health of skin, hair, blood vessels, cartilage, tendons, connective tissue, muscle, bones, teeth and periodontal tissues as described below:

[0035] Skin Health: Collagen plays a vital role in skin health. Collagen I and collagen III are formed in higher proportions in the skin compared to other types of collagen and remain in a fixed ratio in normal skin tissue. Collagen I accounts for about 70% of the collagen in the skin, while collagen III accounts for about 10%. Collagen IV, collagen V, collagen VI, and collagen VII each make up trace amounts of collagen in the skin. Collagen maintains the firmness and elasticity of the skin. Collagen in the form of hydrolyzed collagen helps keep the skin hydrated. As we age, the level of collagen in the body decreases, causing sagging skin, the appearance of fine lines, wrinkles, lack of tone and elasticity, and delayed wound healing.

[0036] Wound Healing: Collagen is a key protein in connective tissue and plays a vital role in wound healing and repairing scarring. Age-related delays in wound healing are due to impaired collagen production and increased degradation.

[0037] Bone: The organic part of bone is about 95% collagen, mainly collagen I. The combination of hard mineral and flexible collagen prevents bones from breaking too easily and makes bones stronger than cartilage. The network of collagen and water forms a strong and smooth pad in the joints, providing cushioning for the ends of bones within the joints during muscle movement.

[0038] Cartilage, tendons, ligaments: Long, thin, fibrous collagen is found primarily in fibrous tissues, such as tendons and ligaments. This flexible and elastic protein is used by the body to support tissues, thus playing an important role in the maintenance of cartilage, tendons, and ligaments. Normal tendons are composed of dense bundles of collagen fibers surrounded by a tendon sheath, forming a soft, fibrous connective tissue. Collagen II is the main component of cartilage.

[0039] Muscle: In muscle tissue, collagen is the main component of the endomysium.

[0040] Tooth tissue: The organic part of dentin and pulp is mainly composed of collagen, mainly collagen I, with small amounts of collagen III and collagen IV. The main collagen found in cementum is collagen I, while in the periodontal ligament it is composed of collagen I, collagen III and collagen XII.

[0041] Basement membrane: The epithelial basement membrane is composed of collagen IV and collagen VII.

[0042] The most common causes of collagen-related disorders are genetic or nutritional defects that affect the biosynthesis, assembly, post-translational modification, secretion or other processes of normal collagen production. The various collagen-related disorders are described below:

[0043] Osteogenesis imperfecta is a dominant autosomal disorder caused by a mutation in collagen I. Osteogenesis imperfecta results in weak bones and irregular connective tissue. In some cases, it can be mild, but in severe cases, it can be fatal. Mild cases are characterized by reduced levels of collagen I, while severe cases are characterized by structural defects in collagen.

[0044] Chondrodysplasia, caused by collagen II mutations, is a bone disorder believed to be the subject of ongoing research.

[0045] Osteoporosis develops with age, is not inherited, and is associated with decreased levels of collagen in the skin and bones. Growth hormone injections are being studied as a potential osteoporosis treatment to offset any loss of collagen.

[0046] Dermal papilla cells are located at the very base of the hair roots in the subcutaneous layer of the scalp and are essential for the development and growth of hair. In practice, new hair growth can occur when dermal papilla cells are harvested from the scalp, cultured, and then transplanted. However, there are several challenges to using dermal papilla cells to treat hair loss. First, it is difficult to isolate these cells from the scalp, the culture conditions are strict, and it is challenging to culture sufficient numbers of cells. In addition, when cultured in large numbers, the hair regeneration ability of these cells decreases significantly. Notably, during the growth phase, dermal papilla cells express a large amount of collagen, but during the regression phase, collagen expression in these cells decreases. Therefore, promoting collagen production in dermal papilla cells can improve hair loss.

[0047] Collagen is also a component of blood vessels and intestines, and its synthesis decreases or deteriorates with age, leading to various diseases. When ascorbic acid is deficient, collagen synthesis is abnormal, resulting in damage to the blood vessel wall, causing scurvy characterized by systemic bleeding. If collagen in the blood vessels (vascular walls) is deficient or deteriorates, the blood vessels lose elasticity, hindering smooth expansion and contraction, leading to high blood pressure. In particular, when collagen in the arterial wall deteriorates, it causes arteriosclerosis, leading to myocardial infarction or cerebral infarction.

[0048] Collagen is necessary for wound healing in the body. It is not only important for the suturing of skin wounds, but also for the healing of internal organ wounds. For the stomach, which is a digestive organ and is often exposed to highly acidic gastric juice, a lack of collagen can easily lead to gastritis, making it difficult for stomach wounds to heal.

[0049] As we age, the lack and degradation of collagen accelerates the aging of all organs, and the reduction of skin collagen is particularly obvious, which is the main cause of skin aging. The skin is composed of three layers: epidermis, dermis and subcutaneous tissue. The dermis is rich in collagen, which plays a role in maintaining skin elasticity. In addition, the stratum corneum and lipid membrane protect the skin surface and retain moisture, but these functions weaken with age. Therefore, as people age, their skin becomes dry, rough and wrinkled. Intake of collagen helps restore skin elasticity and moisture and reduce wrinkles.

[0050] Insufficient collagen in the scalp can lead to hair loss. Although collagen is not a component of the hair itself, the hair root extends to the dermis. Therefore, a lack of collagen in the dermis can hinder healthy hair growth. Conversely, sufficient collagen leads to thicker, healthier hair and an increase in the number of hairs.

[0051] The main component of articular cartilage is collagen. As we age, the loss of collagen weakens the cartilage and makes it unable to absorb impact during joint movement. This causes bones to rub against each other, causing joint inflammation, swelling, fluid accumulation, and eventually degenerative arthritis, accompanied by joint deformity and pain.

[0052] Collagen metabolism is closely related to the development and health of bones and teeth throughout life. Lack of collagen in childhood can limit the development of bones and teeth, as well as affect overall body development. As we age, we are more likely to develop osteoporosis due to insufficient calcium absorption, and collagen deficiency is also an important cause of osteoporosis.

[0053] The main component of the periodontal membrane (periodontal ligament) between the teeth and the alveolar bone (jaw bone) is collagen. Collagen deficiency makes the periodontal membrane and gums fragile, resulting in tooth root exposure, food residue adhesion, and bacterial growth, causing periodontitis.

[0054] The composition comprising amidated amino acids provided by the present invention enhances the body's ability to produce collagen by upregulating the production of collagen and related proteins, thereby improving the health of skin, hair, blood vessels, cartilage, tendons, connective tissue, muscle, bone, teeth and periodontal tissue.

[0055] More specifically, the composition of the present invention can promote collagen production in various cells, including fibroblasts, hair follicle cells, dermal papilla cells, endothelial cells, chondrocytes, myocytes, and osteoblasts.

[0056] Therefore, the composition of the present invention can be characterized in that it is used to prevent or treat diseases or lesions selected from the following: reduced skin elasticity and wrinkles due to aging; hypertension and reduced blood flow due to reduced vascular elasticity; reduced ligament elasticity; reduced joint elasticity; elastic tissue degeneration, dermal-epidermal atrophy, dermal atrophic skin diseases due to photoaging; trauma; burns; skin lesions; pressure sores; skin hypoplasia caused by drug administration; bone and tooth development disorders, osteoporosis; periodontal disease; hair loss.

[0057] The pharmaceutical composition of the present invention comprises amidated amino acids, specifically alaninamide, cysteine, glutamic acid-α-amide, phenylalaninamide, glycineamide, isoleucineamide, lysinamide, leucineamide, methionineamine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valineamide and tyrosinamide, which may be contained in the composition alone or in a combination of two or more.

[0058] In a preferred embodiment of the present invention, the pharmaceutical composition comprises glycineamide, isoleucineamide, leucineamide, methionineamide, prolineamide, valineamide and tyrosinamide, which may be contained in the composition alone or in combination of two or more.

[0059] In the most preferred embodiment of the present invention, the pharmaceutical composition may comprise glycineamide.

[0060] The amide form amino acid of the present invention can be used in its free form or as a pharmaceutically acceptable salt. In the present invention, "pharmaceutically acceptable" means that the substance is physiologically acceptable, does not interfere with the activity of the active ingredient when applied to the human body, and generally does not cause allergic reactions or similar reactions, such as gastrointestinal disorders or dizziness. Preferred salts are acid addition salts formed by pharmaceutically acceptable free acids, and the free acids can be organic acids or inorganic acids. The organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, p-toluenesulfonic acid, glutamic acid and aspartic acid. The inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

[0061] The pharmaceutical composition of the present invention comprising a pharmaceutically acceptable carrier for promoting collagen production can be formulated in various ways according to the route of administration by methods known in the art. The carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microparticles.

[0062] The pharmaceutical composition of the present invention can be administered to a patient in an amount that effectively promotes collagen production. For example, a typical daily dose can be about 0.01 to 1000 mg / kg, and preferably about 50 to 500 mg / kg. The pharmaceutical composition of the present invention can be administered in a single dose or divided dose within a preferred dosage range. In addition, those skilled in the art can appropriately select the dosage of the pharmaceutical composition of the present invention according to route of administration, subject of administration, age, sex, body weight, individual differences and disease condition.

[0063] The administration route may be oral or parenteral. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intraosseous, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration.

[0064] When the pharmaceutical composition of the present invention is orally administered, it can be formulated into dosage forms such as powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, thin tablets, etc. together with appropriate oral administration carriers according to methods known in the art. Examples of suitable carriers include sugars, including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; starches, including corn starch, wheat starch, rice starch, potato starch; celluloses, including cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose; fillers, such as gelatin and polyvinyl pyrrolidone. In addition, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate can be added as disintegrants as needed. In addition, the pharmaceutical composition can also contain anti-caking agents, lubricants, humectants, flavoring agents, emulsifiers, preservatives, etc.

[0065] In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated into injection, transdermal delivery system and nasal inhalation forms together with suitable parenteral carriers using methods well known in the art. For injection, it must be sterile and should be prevented from being contaminated by microorganisms such as bacteria and fungi. Suitable carriers for injection can include, but are not limited to, solvents or dispersion media, including water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers can include isotonic solutions, such as Hank's solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% glucose. In order to prevent injection from being contaminated by microorganisms, various antibacterial and antifungal agents can be included in addition, such as parabens, chlorobutanol, phenol, sorbic acid and thimerosal. In addition, in most cases, the injection can also include isotonic agents, such as sugar or sodium chloride.

[0066] In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, pastes, liniments and aerosols are included. "Transdermal administration" means that an effective amount of active ingredients contained in the pharmaceutical composition is delivered to the skin by applying the pharmaceutical composition topically to the skin. For example, the pharmaceutical composition of the present invention can be made into an injectable form and applied by gently piercing the skin with a fine 30-gauge needle or directly applying it to the skin. These formulations are described in the formulas generally known in pharmaceutical chemistry.

[0067] In the case of administration by inhalation, the compounds for use according to the invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0068] Other pharmaceutically acceptable carriers may refer to those known in the art.

[0069] In addition, the pharmaceutical compositions of the present invention may also contain one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents and / or preservatives.

[0070] Furthermore, the pharmaceutical compositions of the present invention can be formulated by methods known in the art so as to provide quick, sustained or delayed release of the active ingredient after administration to a mammal.

[0071] Furthermore, the pharmaceutical composition of the present invention may be administered alone or in combination with a known compound having an effect of promoting collagen production.

[0072] In one aspect of the present invention, the known compound may include ascorbic acid, asiatica saponin, glucosamine, or analogs and derivatives thereof.

[0073] In the present invention, the known compound may be administered in combination with an amidated amino acid in a molar ratio of amidated amino acid:known compound=1:0.1-5.

[0074] In one embodiment of the present invention, it has been demonstrated that the combination treatment of amidated amino acids with ascorbic acid, asiatica saponin, glucosamine or the like achieves a synergistic or cooperative effect in promoting intracellular collagen production.

[0075] In the present invention, the ascorbic acid analogue may be selected from magnesium ascorbyl phosphate, 3-O-ethyl ascorbic acid, ascorbyl glucoside and ascorbyl tetraisopalmitate.

[0076] The present invention also provides a cosmetic composition for promoting collagen production, which comprises one or more amidated amino acids selected from alanineamide, cysteine, glutamic acid-α-amide, phenylalanineamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionine, amidated asparagine, prolineamide, amidated glutamine, arginineamide, threonineamide, valineamide and tyrosinamide.

[0077] The cosmetic composition can be used to improve skin wrinkles, enhance skin moisture, improve skin elasticity or prevent skin aging.

[0078] The cosmetic composition of the present invention may include one amidated amino acid selected from the group of amidated amino acids, or may include a combination of two or more amidated amino acids selected from the group of amidated amino acids.

[0079] The cosmetic composition of the present invention can be prepared in any formulation generally produced in the art, and it can also be formulated as an adjuvant for topical or systemic application commonly used in the dermatology field by containing a dermatologically acceptable medium or base in addition to the amidated amino acid.

[0080] In addition, the cosmetic composition of the present invention may further contain, in addition to the amidated peptide, adjuvants commonly used in the field of beauty or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion complexing agents and chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics. Moreover, these ingredients can be introduced in the amounts commonly used in the field of dermatology.

[0081] Suitable formulations of the cosmetic composition may include, for example, solutions, gels, solid or pasty anhydrous products, emulsions, suspensions, microemulsions, microcapsules, microparticles or vesicle dispersions in ionic (liposomes) or non-ionic forms obtained by dispersing the oil phase in the aqueous phase, creams, skin care lotions, powders, ointments, sprays or concealer sticks. In addition, the cosmetic composition may also be made into the form of foam or aerosol compositions, further comprising a compressed propellant. The products to which the cosmetic composition of the present invention may be added include, but are not limited to, skin care lotions, essences, nutritional essences, cosmetic bags, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, care products, beauty serums, lotions, pressed powders, loose powders and eye shadows.

[0082] The content of the amide-modified amino acid contained in the cosmetic composition of the present invention comprises 0.0001 to 50% by weight, preferably 0.01 to 10% by weight, based on the total weight of the cosmetic composition, and can be appropriately determined by those skilled in the art based on factors such as the desired effect, degree of application, type of preparation, and stability of the peptide in the cosmetic composition.

[0083] The present invention also provides a food composition for promoting collagen production, which comprises one or more amidated amino acids selected from the following: alaninamide, cysteine, glutamate-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0084] The food composition is characterized in that it is used to prevent or improve the following diseases or lesions: reduced skin elasticity and wrinkles due to aging; hypertension and poor blood circulation due to reduced vascular elasticity; reduced ligament elasticity; reduced joint elasticity; elastic tissue degeneration, dermal-epidermal atrophy, dermal atrophic skin diseases due to photoaging; trauma; burns; skin lesions; pressure sores; skin hypoplasia caused by drug administration; bone and tooth development disorders, osteoporosis, periodontal disease and hair loss.

[0085] Food composition of the present invention includes all forms, for example functional food, nutritional supplement, health food and food additive.These types can be prepared in various forms according to conventional methods known in the art.In order to use food composition of the present invention in the form of food additive, it can be prepared into powder or concentrate and used.

[0086] Meanwhile, the food composition of the present invention may include one amidated amino acid selected from the group of amidated amino acids, or may include a combination of two or more amidated amino acids selected from the group of amidated amino acids.

[0087] As health food, the food composition of the present invention can be eaten by preparing it into the form of tea, juice or beverage, or by making it into granules, capsules or powders. In addition, the food composition of the present invention can be prepared with known substances or active ingredients that are known to promote collagen generation.

[0088] Functional foods can be produced by adding the food composition of the present invention to beverages (including alcoholic beverages), fruits and processed products thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meat and processed products thereof (e.g., ham, sausages, corned beef, etc.), bread and noodles (e.g., udon noodles, soba noodles, ramen noodles, pasta, macaroni, etc.), fruit juices, various beverages, biscuits, toffees, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, steamed foods, frozen foods, and various condiments (e.g., soybean paste, soy sauce, sauces, etc.).

[0089] The preferred content of the peptide in the food composition of the present invention (but not limited thereto) is preferably 0.01 to 50% by weight of the final food.

[0090] The present invention provides the use of one or more amidated amino acids selected from the following in the preparation of a composition for promoting collagen production: alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine and tyrosinamide.

[0091] The present invention provides a method for promoting collagen production, comprising administering to a subject in need thereof an effective amount of one or more amidated amino acids selected from the group consisting of alanineamide, cysteine, glutamic acid-α-amide, phenylalanineamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valineamide and tyrosinamide.

[0092] The term "effective amount" in the present invention refers to an amount that exhibits the effect of promoting collagen production when administered to a subject, wherein the "subject" may be an animal, preferably a mammal, especially an animal including a human, or a cell, tissue, organ, etc. derived from an animal. The subject may be a patient who needs the above-mentioned effect.

[0093] In this specification, the term "comprising" has the same meaning as "including" or "characterized by", and in the context of the composition or method according to the present invention, it does not exclude other components or method steps not explicitly mentioned. In addition, the term "consisting of..." means excluding any other elements, steps or components not explicitly stated. The term "consisting essentially of..." means that the composition or method may include substances or steps that do not substantially affect the basic characteristics of the listed substances or steps.

[0094] Effects of the Invention

[0095] The composition of the present invention comprising amidated amino acids is very useful for developing treatment methods for various diseases and pathologies that may be caused by collagen deficiency by promoting collagen production in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1a and 1b Shown are the results of evaluating the effects of 20 amidated amino acids on collagen production and cell viability in human skin fibroblasts.

[0098] Figure 2a and 2b Shown are the results of evaluating the effects of 20 free amino acids on collagen production and cell viability in human skin fibroblasts.

[0099] Figures 3a to 3c Shown are the results of evaluating the effects of glycine or its analogs on collagen production and cell viability in human skin fibroblasts.

[0100] Figures 4a to 4d Concentration-dependent effects of ascorbic acid (AA), glycine, and glycineamide on collagen production and cell viability in human skin fibroblasts are shown.

[0101] Figure 5a and 5b Shown are the results of evaluating the effects of ascorbic acid (AA), glycinamide, and TGF-β1 treatment alone or in combination on collagen production and cell proliferation in human skin fibroblasts.

[0102] Figure 6a and 6b Shown are the results of evaluating the effects of ascorbic acid (AA), glycinamide, and TGF-β1 treatment alone or in combination on wound healing of human dermal fibroblasts.

[0103] Figures 7a to 7c Shown are the results of evaluating the effects of ascorbic acid (AA), its analogs, glycinamide, and TGF-β1 treatment alone or in combination on collagen production and cell proliferation in human dermal fibroblasts.

[0104] Figure 8a and 8b Shown are the results evaluating the effects of ascorbic acid (AA), its analogs, glycinamide, and TGF-β1 treatment alone or in combination on wound healing of human dermal fibroblasts.

[0105] Figure 9a and 9b Shown are the results of evaluating the effects of asiaticoside, madecassoside, asiatic acid, and madecassic acid on cell viability and collagen production in human skin fibroblasts.

[0106] Fig.10a and 10b Shown are the results of evaluating the effects of asiatica saponin, madecassoside, and ascorbic acid on cell viability and collagen production in human skin fibroblasts.

[0107] Figures 11a and 11b show the results of evaluating the effects of asiatica saponin and glycineamide alone or in combination on the viability and collagen production of human skin fibroblasts.

[0108] Figures 12a to 12j Shown are the results of evaluating the effects of glycine (G), proline (P), lysine (K) or their amidated amino acids (G-NH2, P-NH2, K-NH2) on collagen production and cell proliferation (MTT reduction) in human articular chondrocytes (HAC), human dermal fibroblasts (HDF), human hair follicle dermal papilla cells (HFCPC), human aortic endothelial cells (HAoEC) and human osteoblasts (HOB).

[0109] Figures 13a to 13l Concentration-dependent effects of glucosamine (GlcN), glycinamide (G-NH2), and TGF-β1 treatment on collagen production and cell proliferation (MTT reduction) in human dermal fibroblasts and human articular chondrocytes are shown.

[0110] Figures 14a to 14d Shown are the results evaluating the effects of glucosamine (GlcN) and glycinamide (G-NH2) treatment alone or in combination on collagen production and cell proliferation (MTT reduction) in human articular chondrocytes and human skin fibroblasts.

[0111] Detailed description of implementing the invention

[0112] The present invention will be described in detail below in conjunction with the following embodiments. However, the following embodiments are only examples of the present invention, and the present invention is not limited thereto.

[0113] Experimental methods

[0114] 1 Experimental samples

[0115] Twenty free amino acids and 20 amidated amino acids were used in this study. These compounds are listed in Table 1 with the single letter code of the amino acid. Free amino acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amidated amino acids were purchased from Watanabe Chemical Ind., Ltd. (Hiroshima, Japan). N-acetylglycine, N-acetylglycinamide, glycine methyl hydrochloride, glycine ethyl hydrochloride, glycylglycine, ascorbic acid (AA), 3-O-ethyl ascorbic acid (EAA) and ascorbic acid 2-O-glucoside (AG), glucosamine hydrochloride were purchased from Sigma-Aldrich. Magnesium ascorbyl phosphate (MAP) and ascorbyl tetraisopalmitate (ATI) were purchased from Biosynth Carbosynth (Berkshire, UK). Recombinant human TGF-β1 protein was purchased from R&D Systems (Minneapolis, MN, USA). Asiaticosaponin, madecassoside, asiatic acid and madecassic acid were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

[0116] [Table 1]

[0117]

[0118] 2. Cell Culture

[0119] Human dermal fibroblasts (HDF) extracted from adult human skin were obtained from Cascade Biologics (Portland, OR, USA), and human articular chondrocytes (HAC), human hair follicle dermal papilla cells (HFCPC), human aortic endothelial cells (HAoEC), and human osteoblasts (HOB) were obtained from PromoCell (Heidelberg, Germany). The cells were cultured in a closed incubator at 37°C in a humidified atmosphere containing 5% CO2. The growth medium for HDFs consisted of Iscove's modified Dulbecco's medium (IMDM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT) and 1% antibiotics (100 U mL -1 Penicillin, 0.1 mg mL -1HACs were cultured in chondrocyte growth medium, HFCPCs were cultured in hair follicle dermal papilla cell growth medium, HAoECs were cultured in endothelial cell medium MV, and HOBs were cultured in osteoblast growth medium (PromoCell, Heidelberg, Germany), all of which were supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT) and 1% antibiotics (100 U mL -1 Penicillin, 0.1 mg mL -1 Streptomycin).

[0120] 3. Cell Viability Assay

[0121] Cell viability was assessed using the MTT assay. Briefly, HDFs (1 × 10 4 cells), HAC (5×10 3 cells), HFDPC (1×10 4 cells), HAoEC (5×10 3 cells per well) and HOB (2×10 3 cells) in growth medium in 96-well culture plates for 24 h and then treated with each drug for 48 h. After discarding the medium, the cells were incubated in a 1 mg mL -1 MTT (Amresco, Solon, OH, USA) in fresh growth medium (100 μL) at 37 ° C for 2 hours. The medium was removed by aspiration, and the intracellular accumulated formazan dye was dissolved in 100 μL dimethyl sulfoxide. The absorbance of the solution was measured at 570 nm using a Spctrostar Nano microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

[0122] 4. Collagen Measurement

[0123] Sircol was used according to the manufacturer's instructions. TM Collagen levels in conditioned medium were assessed using the collagen assay (Biocolor, Antrim, UK). HDFs (2 × 10 5 cells per well), HAC (3×10 5 cells), HFDPC (2×10 5 cells per well), HAoEC (3×10 5 cells per well) and HOB (3×10 5Cells (100 μL) were cultured in growth medium in 12-well culture plates for 24 hours and then treated with each drug for an additional 24 hours. After discarding the medium, the cells were washed with 500 μL of phosphate-buffered saline (PBS) and then incubated in 1 mL of serum-free medium for another 24 hours. 1.0 mL of the medium sample (conditioned medium or fresh medium as a negative control) was transferred to a microcentrifuge tube, mixed with 100 μL of collagen separation and concentration reagent, and incubated overnight at 4°C. The samples were centrifuged at 13,000 × g for 10 minutes at 4°C, and the supernatant was carefully removed with a pipette without disturbing the precipitate. 500 μL of Sircol dye reagent was then added to each tube, inverted five times, and shaken at room temperature for 30 minutes. After centrifugation, the supernatant was discarded and the precipitate was washed with 500 μL of cold saline wash reagent, followed by another centrifugation step. The precipitate was then dissolved in 250 μL of alkaline reagent, and a 200 μL portion was transferred to a 96-well plate. The optical density of the solution was read at 555 nm using a Spectrostar Nano microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

[0124] 5. Wound Healing Assay

[0125] Oris was used according to the manufacturer's instructions. TM Wound closure assay was performed using the Universal Cell Migration Assembly Kit (Platinus Technologies, WI, USA). TM Plug Insertion with Oris TM A suspension of human dermal fibroblasts in IMDM was added to each well of a compatible 96-well plate (100 μL per well, 1 × 10 4 Cells) and incubated for 12 hours to allow cells to attach. Then, the plug was removed using a plug tool, and the culture medium was aspirated using a pipette. The wells were gently washed with 100 μL PBS to remove unattached cells, and the attached cells were treated with drugs in 100 μL culture medium for a specified time. Wound images were acquired using a phase contrast microscope (Eclipse TS100, Nikon Instruments Inc., Melville, NY, USA). The wound area in the image was quantified using the NIHImage J program. The wound closure rate (%) was calculated using the following formula: wound closure rate (%) = [(A0-At) / A0] × 100, where A0 is the original wound area, and At is the wound area measured at the specified time.

[0126] Experimental Results

[0127] 1. Effects of amidated amino acids on collagen production in human skin fibroblasts

[0128] In the first experiment, all 20 C-terminally amidated amino acids corresponding to naturally occurring free amino acids were compared with ascorbic acid. Cells were treated with 1 mM of each amidated amino acid or ascorbic acid. After 48 hours, the conditioned medium was harvested and used to quantify collagen by colorimetric assay. Cell viability was assessed using the MTT method for adherent cells. Figure 1a As shown in the figure, various amino acids can enhance the level of collagen secreted into the culture medium, among which glycineamine (G-NH2) has the most obvious effect, followed by leucineamide (L-NH2), tyrosinamide (Y-NH2), valineamide (V-NH2), prolineamide (P-NH2), methionine (M-NH2), isoleucineamide (I-NH2), phenylalanineamide (F-NH2), alanineamide (A-NH2), amidated glutamine (Q-NH2), cysteine ​​(C-NH2), threonine (T-NH2), glutamic acid α-amide (E-NH2) and arginine amine (R-NH2). However, other amidated amino acids such as amidated asparagine (N-NH2), lysinamine (K-NH2), serine (S-NH2), tryptophanamine (W-NH2), aspartic acid α-amide (D-NH2) and histidine (H-NH2) have no obvious effect. Effects on cell viability are shown in Figure 2. Figure 1b As shown, lysineamide and aspartic acid α-amide showed a decreasing effect, while other amidated amino acids had no significant effect. Therefore, glycineamide was identified as the most effective in enhancing cellular collagen production.

[0129] 2. Effects of free amino acids on collagen production in human skin fibroblasts

[0130] In the following experiments, all 20 naturally occurring free amino acids were compared with ascorbic acid. Cells were treated with 1 mM of each amino acid or ascorbic acid for 48 h. Figure 2a As shown, among the free amino acids, glycine (G) was the most effective in increasing collagen, followed by proline (P), isoleucine (I), glutamine (Q) and leucine (L), while other amino acids did not increase collagen. In this experiment, glycine amide (G-NH2) was also compared, and it was found that glycine amide had a superior collagen-promoting effect compared to all free amino acids. In the cell viability experiment, various amino acids had minimal effects on cell viability ( Figure 2b ).

[0131] 3. Effects of glycine or its analogs on collagen production in human skin fibroblasts

[0132] In the following experiments, various glycine analogs ( Figure 3a ) on cellular collagen production. When cells were treated with each glycine analog at a concentration of 1 mM, glycine amide showed the highest collagen production-promoting effect, with a significant improvement compared to glycine ( Figure 3b ). Other glycine analogs, such as N-acetylglycine, glycine ethyl ester, and glycylglycine, did not affect collagen production, while N-acetylglycine amide and glycine methyl ester had a certain reducing effect. All glycine analogs did not significantly affect cell viability ( Figure 3c ).

[0133] 4. Concentration-dependent effects of ascorbic acid, glycine, and glycinamide

[0134] In the following experiment, the effects of ascorbic acid, glycine, and glycylglycine on collagen production were compared according to their concentrations. Figure 4a and 4b As shown, ascorbic acid increased collagen production at 1 mM without affecting cell viability, but significantly reduced cell viability and collagen production at 3 and 5 mM. Glycine at 1 mM enhanced collagen production without affecting cell viability, but at 3 or 5 mM, it significantly reduced cell viability and collagen production. Glycylglycine at 1 mM promoted collagen production without affecting cell viability, but at 3 or 5 mM, it slightly reduced cell viability and collagen production. Figure 4c and 4d As shown, glycylglycine increased collagen production even at a low concentration of 0.25 mM and exhibited the greatest enhancement of collagen production without reducing cell viability at 1-2 mM.

[0135] 5. Effects of ascorbic acid, glycinamide, and TGF-β1 alone or in combination on collagen production in human skin fibroblasts

[0136] Since ascorbic acid and glycinamide were observed to promote collagen production individually, further studies were performed to determine whether their combined treatment exhibited a synergistic effect in the absence and presence of TGF-β1. Figure 5a As shown, in the absence of TGF-β1, ascorbic acid (1 mM) and glycine amide (1 mM) increased collagen production by 23.1% and 68.2%, respectively, compared with the control group. The combined treatment increased collagen production by 153% compared with the control group, which was significantly higher than the arithmetic sum of the individual effects (91.3%). This experimental result shows that the combined treatment of ascorbic acid and glycine amide synergistically promoted collagen production. As expected, TGF-β1 (10 ng mL -1) itself showed an effect of enhancing collagen production. The combined treatment of ascorbic acid and glycine amide increased collagen production to a similar extent as TGF-β1. The enhancing effect of ascorbic acid and glycine amide alone or in combination on collagen production was greater in the absence of TGF-β1 than in the presence of TGF-β1. Figure 5b As shown in Figure 2, TGF-β1 significantly increased the number of viable cells by 44.5% compared with the control group. When treated with ascorbic acid and glycine amine alone, cell viability did not change significantly; however, their combined treatment significantly increased the number of viable cells by 14.8% compared with the control group. This result also shows that the combined treatment of ascorbic acid and glycine amine has a synergistic effect in promoting cell proliferation.

[0137] 6. Effects of ascorbic acid, glycinamide, and TGF-β1 on wound healing in human skin fibroblasts

[0138] In the following experiments, it was investigated whether ascorbic acid, glycinamide, and TGF-β1 treatment alone or in combination could promote wound closure. Cells were cultured in wells with plugs to create circular wounds. After the plugs were removed, cells were treated with each drug for up to 48 hours to monitor wound closure over time. Figure 6a Representative microscopic images of wounds before and after treatment are shown. Initially, wound extent was the same in all groups, but the extent of wound closure varied among treatments. Relative wound closure was quantified by image analysis and the results are shown in Figure 6b As shown. After 12 hours of treatment, ascorbic acid or glycine amide significantly improved wound closure compared with the control group, and the combined treatment further enhanced the effect to a level similar to that of the TGF-β1 treated group. The change caused by the combined treatment of ascorbic acid and glycine amide (32.1%) is much greater than the arithmetic sum of the changes caused by the individual treatments (2.5% + 6.3% = 8.8%). These experimental results show that the combined treatment of ascorbic acid and glycine amide can synergistically promote wound closure. The synergistic effect of the combined treatment of ascorbic acid and glycine amide was also observed 24 hours after treatment (100% >> 9.1% + 19.2%). Even in the presence of TGF-β1, the combined treatment of ascorbic acid and glycine amide further enhanced wound closure. These results show that the combined treatment of ascorbic acid and glycine amide is as effective as TGF-β1 in promoting wound closure.

[0139] 7. Effects of various ascorbic acid analogs, glycinamide, and TGF-β1 on collagen production in human skin fibroblasts

[0140] To investigate the effects of various ascorbic acid analogs on collagen production ( Figure 7a), cells were treated with each analog alone or in combination with glycinamide and / or TGF-β1, and collagen production and cell viability were assessed. Ascorbic acid analogs, such as magnesium ascorbyl phosphate (MAP), 3-O-ethylascorbic acid (EAA), ascorbic acid 2-O-glucoside (AG), and ascorbyl tetraisopalmitate (ATI), significantly enhanced collagen production to a similar extent as ascorbic acid ( Figure 7b ). When each ascorbic acid analog was combined with glycineamide, a synergistic effect on collagen production was observed. The changes caused by the combined treatment of ascorbic acid analogs with glycineamide (MAP, 118.4%; EAA, 115.5%; AG, 123.6%; ATI, 124.6%) were greater than the arithmetic sum of the changes caused by the individual treatments of ascorbic acid analogs and glycineamide (MAP, 25.3% + 67.4% = 92.7%, EAA, 19.5% + 67.4% = 86.9%, AG, 27.2% + 67.4% = 94.6%, ATI, 25.5% + 67.4% = 92.9%). These experimental results show that the combined treatment of ascorbic acid analogs and glycineamide can synergistically promote collagen production. These results show that specific ascorbic acid analogs can be used in combination with glycineamide to enhance collagen production. Cell viability was not significantly affected when each ascorbic acid analog was used alone or in combination with glycinamide; however, some ascorbic acid analogs (EAA, AG, and ATI) significantly reduced the cell proliferation effect of TGF-β1 ( Figure 7c ).

[0141] 8. Effects of various ascorbic acid analogs, glycinamide, and TGF-β1 on wound healing in human skin fibroblasts

[0142] The effects of various ascorbic acid analogs on wound closure in the absence or presence of glycinamide were compared. For comparison, the effects of TGF-β1 were tested. To monitor wound closure over time, cells were treated with vehicle or drug for 48 hours. Initially, the extent of wounds was the same in all groups, but the extent of wound closure varied by treatment ( Figure 8a ). Relative wound closure was quantified by image analysis and the results are shown in Figure 8bTGF-β1 significantly improved wound closure compared with the control group at 12 and 24 h after treatment. The effects of ascorbic acid analogs on wound closure showed various patterns. Magnesium ascorbyl phosphate (MAP) promoted wound closure to a similar extent as ascorbic acid, and glycinamide further enhanced its effect. Other analogs, such as 3-O-ethylascorbic acid (EAA), ascorbic acid 2-O-glucoside (AG), and ascorbyl tetraisopalmitate (ATI), slightly promoted wound closure in the early stage (24 h) but inhibited wound closure in the later stages (36 and 48 h). Treatment with glycinamide alone promoted wound closure, an effect that was further enhanced by combined treatment with magnesium ascorbyl phosphate (MAP), but not with 3-O-ethylascorbic acid (EAA), ascorbic acid 2-O-glucoside (AG), or ascorbyl tetraisopalmitate (ATI). These results suggest that each ascorbic acid analog has a different effect on wound closure and that glycinamide promotes wound closure under a variety of conditions in the absence or presence of an ascorbic acid analog or TGF-β1.

[0143] 9. Effects of triterpenoids from Centella asiatica L. on collagen production in human skin fibroblasts

[0144] The wound healing effect of Centella asiatica extract is related to collagen production, angiogenesis improvement and inflammation inhibition. It is reported that its main pharmacologically active ingredients are triterpenoids, such as Centella asiatica saponin, hydroxy Centella asiatica saponin, asiatic acid and hydroxy Centella asiatica acid. In the following experiment, the effects of these four ingredients on cell viability and collagen production were compared ( Figure 9a , 9b ). When cells were treated with 10 μM of each compound, both asiatic acid and madecassic acid reduced cell viability and collagen production, while madecassinoside had no significant effect on either. In contrast, asiatica saponin significantly increased collagen production without affecting cell viability.

[0145] 10. Comparison of the effects of Centella asiatica saponin, hydroxy Centella asiatica saponin and ascorbic acid on collagen production in human skin fibroblasts

[0146] In the following experiments, the effects of various concentrations of asiatica saponin, madecassoside, and ascorbic acid on cell viability and collagen production were evaluated ( Fig.10a , 10b). Asiatic acid and madecassic acid had no effect on cell viability at concentrations below 100 μM, but reduced cell viability at concentrations above 300 μM, while ascorbic acid also had no effect on cell viability at concentrations up to 1000 μM. Therefore, the effects of these compounds on collagen production at non-cytotoxic concentrations were compared. Madecassosaccharin increased collagen production at a concentration of 100 μM, asiatica saponin increased collagen production at concentrations of 10-100 μM, and ascorbic acid increased collagen production at concentrations of 10-1000 μM. The effect of 100 μM asiatica saponin was statistically comparable to the effect of 1000 μM ascorbic acid.

[0147] 11. Effects of Centella asiatica saponins and glycinamide alone or in combination on collagen production in human skin fibroblasts

[0148] The cell viability and collagen production were compared when asiatic saponin and glycine amide were treated alone or in combination (Figures 11a, 11b). The change caused by the combined treatment of 100 μM asiatic saponin and 100 μM glycine amide (131.7%) was significantly greater than the arithmetic sum of the changes caused by the individual treatments of 100 μM asiatic saponin and 100 μM glycine amide (51.8% + 27.2% = 79.0%). The change caused by the combined treatment of 100 μM asiatic saponin and 1000 μM glycine amide (218.7%) was significantly greater than the arithmetic sum of the changes caused by the individual treatments of 100 μM asiatic saponin and 1000 μM glycine amide (51.8% + 75.7% = 127.5%). This result shows that the combined treatment of asiatic saponin and glycine amide can synergistically promote the production of collagen in cells.

[0149] 12. Effects of glycine, proline, lysine or their amidated amino acids on collagen production in various cells

[0150] To investigate the effects on collagen production in various cells, human articular chondrocytes (HAC), human hair follicle dermal papilla cells (HFCPC), human aortic endothelial cells (HAoEC) and human osteoblasts (HOB) were treated with 1 mM concentrations of glycine (G), proline (P), lysine (K) and their amidated amino acids glycinamide (G-NH2), prolineamide (P-NH2) and lysineamide (K-NH2). Figures 12a to 12j As shown in Figure 2, although there was no significant difference in cell viability, amidated amino acids showed better collagen production in various cells compared with free amino acids, among which glycine amide was the most effective. It is worth noting that the collagen production effect of glycine amide in HOB was 433.8%. Therefore, it was confirmed that glycine amide is the most effective in enhancing collagen production in various cells.

[0151] 13. Concentration-dependent effects of TGF-β1, glycineamine and glucosamine on human skin fibroblasts and human articular chondrocytes

[0152] The effects of TGF-β1, glycinamide (G-NH2), and glucosamine (GlcN) on cell proliferation and collagen production were compared in two cell types. Figures 13a to 13f As shown, TGF-β1 increased the proliferation of human skin fibroblasts in a concentration-dependent manner, while glycine amine and glucosamine did not affect cell proliferation. As expected, TGF-β1 showed an enhancing effect on collagen production that exceeded its cell proliferation effect. Glycine amine increased collagen production in a concentration-dependent manner without affecting cell proliferation. Similarly, glucosamine increased collagen production without affecting cell proliferation.

[0153] like Figures 13g to 13l As shown in Figure 2, 0.5 mM glucosamine increased collagen production in human articular chondrocytes without affecting cell viability. Glycine amine and TGF-β1 slightly but significantly increased cell viability compared to the control group, with glycine amine enhancing collagen production at a concentration of 0.5 mM and the most significant effect at a concentration of 2 mM, with an increase of 238.6%. TGF-β1 increased collagen production in a dose-dependent manner up to a dose of 10 ng mL -1 .

[0154] 14. Effects of glycine amine, glucosamine and TGF-β1 alone or in combination on collagen production in human articular chondrocytes and human skin fibroblasts

[0155] Since both glycinamide (G-NH2) and glucosamine (GlcN) have been observed to promote collagen production, we further investigated whether their combined treatments in the absence and presence of TGF-β1 would exhibit synergistic effects. Figures 14a to 14d As shown, in the absence of TGF-β1, glycine amine (0.5 mM) and glucosamine (0.5 mM) increased collagen production in human articular chondrocytes by 181.3% and 147.6%, respectively, compared with the control group, and their combined treatment increased collagen production in a concentration-dependent manner. In human skin fibroblasts, glycine amine (0.5 mM) and glucosamine (0.5 mM) increased collagen production by 139.2% and 118.6%, respectively, compared with the control group, and their combined treatment increased collagen production in a concentration-dependent manner. These experimental results indicate that the combined treatment of glycine and glucosamine can synergistically promote collagen production. TGF-β1 alone (10 ng mL -1) showed the effect of enhancing collagen production in human articular chondrocytes and human skin fibroblasts. -1 ), the combined treatment of glycine amine (0.5 mM) and glucosamine (0.5 mM) had a better effect on collagen production in human articular chondrocytes. In the absence of TGF-β1, the collagen production promoting effect of glycine amine and glucosamine alone or in combination was greater than that in the presence of TGF-β1. In human skin fibroblasts and human articular chondrocytes, the combined treatment of glycine amine and glucosamine and the TGF-β1 treatment obtained better cell proliferation effect than the control group.

[0156] Industrial Applicability

[0157] The composition comprising an amidated amino acid of the present invention has high industrial applicability because it can be very useful for developing treatment methods for various diseases and pathologies that may be caused by collagen deficiency by promoting the production of collagen in cells.

Claims

1. A pharmaceutical composition for promoting collagen production, comprising one or more amidated amino acids selected from the group consisting of alanineamide, cysteine, glutamic acid-α-amide, phenylalanineamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionine, amidated asparagine, prolineamide, amidated glutamine, arginineamide, threonineamide, valineamide, and tyrosinamide.

2. The pharmaceutical composition according to claim 1, wherein the composition promotes collagen production in cells.

3. The pharmaceutical composition according to claim 2, wherein the cells are selected from the group consisting of fibroblasts, hair follicle cells, dermal papilla cells, endothelial cells, chondrocytes, myocytes, and osteoblasts.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a composition for preventing or treating a disease or lesion selected from the group consisting of: decreased skin elasticity and wrinkles caused by aging; hypertension and decreased blood flow caused by decreased vascular elasticity; decreased ligament elasticity; decreased joint elasticity; elastic tissue degeneration, dermal-epidermal atrophy, and dermal atrophic skin diseases caused by photoaging; trauma; burns; skin lesions; pressure sores; skin hypoplasia caused by drug administration; bone and tooth development disorders, osteoporosis; periodontal disease; and hair loss.

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises at least one selected from the group consisting of ascorbic acid, asiatica saponin, glucosamine, and analogs and derivatives thereof.

6. The pharmaceutical composition according to claim 5, wherein the ascorbic acid analog is selected from the group consisting of ascorbic acid, ascorbate, magnesium ascorbyl phosphate, 3-O-ethyl ascorbic acid, ascorbyl glucoside, and ascorbyl tetraisopalmitate.

7. The pharmaceutical composition according to claim 5, wherein the asiaticosaponin analog is selected from the group consisting of asiaticosaponin, asiaticosaponin salts, madecassoside, asiatic acid, and madecassoside.

8. A cosmetic composition for promoting collagen production, comprising one or more amidated amino acids selected from the group consisting of alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine, and tyrosinamide.

9. The cosmetic composition according to claim 7, wherein the cosmetic composition is a composition for improving skin wrinkles, enhancing skin moisture, improving skin elasticity, or preventing skin aging.

10. A food composition for promoting collagen production, comprising one or more amidated amino acids selected from the group consisting of alanineamide, cysteine, glutamic acid-α-amide, phenylalanineamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionineamine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valineamide, and tyrosinamide.

11. The food composition of claim 9, wherein the food composition is a functional food composition.

12. Use of one or more amidated amino acids selected from the group consisting of alanineamide, cysteine, glutamic acid-α-amide, phenylalanineamide, glycineamide, isoleucineamide, lysineamide, leucineamide, methionineamine, amidated asparagine, prolineamide, amidated glutamine, arginineamine, threonineamide, valineamide, and tyrosinamide in the preparation of a composition for promoting collagen production.

13. The use according to claim 12, wherein the composition is used to promote collagen production in cells.

14. A method for promoting collagen production, comprising administering to a subject in need thereof an effective amount of a composition comprising one or more amidated amino acids selected from the group consisting of alaninamide, cysteine, glutamic acid-α-amide, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, amidated asparagine, prolineamide, amidated glutamine, arginine, threonine, valine, and tyrosinamide.

15. The method of claim 14, wherein the composition is used to promote collagen production in cells.

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