COMPOSITION FOR PROMOTING BIOSYNTHESIS OF ELASTIN AND COLLAGEN IN CONNECTIVE TISSUE

Abstract

Disclosed is a composition for promoting biosynthesis of elastin or collagen. The composition improves the ability of fibroblasts to biosynthesize elastin and collagen and is thus used to restore skin elasticity deteriorated due to aging or light exposure, remove wrinkles and facial lines, improve vascular elasticity deteriorated due to aging, and hypertension and decreased blood circulation caused thereby, and enhance tendon and ligament elasticity and joint elasticity.

Claims

1. A composition for promoting biosynthesis of elastin or collagen comprising glycine, L-proline and a divalent metal ion, wherein a content ratio of glycine to L-proline is 1:0.9 to 1:1.1.

2. The composition according to claim 1, wherein the composition further comprises one or more amino acids selected from the group consisting of L-alanine, L-valine, L-leucine and L-lysine hydrochloride, and a content ratio of each amino acid to glycine based on weight is as follows: L-alanine: 0.40 to 0.90; L-valine: 0.30 to 0.80; L-leucine: 0.10 to 0.30; L-lysine hydrochloride: 0.10 to 0.25.

3. The composition according to claim 1, wherein the composition further comprises 0.1 to 30.0% by weight of L-cysteine or N-acetyl-L-cysteine.

4. The composition according to claim 1, wherein the divalent metal is selected from the group consisting of copper (Cu), cobalt (Co), calcium (Ca), magnesium (Mg), manganese (Mn) and zinc (Zn).

5. The composition according to claim 4, wherein the divalent metal is copper (Cu).

6. The composition according to claim 4, wherein the divalent metal is present in an amount of 0.001 to 5.0% by weight based on a total weight of the composition.

7. The composition according to claim 1, wherein the composition further comprises hyaluronic acid or a salt thereof.

8. A pharmaceutical composition for promoting biosynthesis of elastin or collagen comprising the composition according to claim 1.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is formulated as an oral administration form, an intramuscular injection form, a percutaneous injection form, an external medicine form or a medical device.

10. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is used for prevention or treatment of elastin-related diseases, hypertension due to decreased vascular elasticity, decreased blood circulation, decreased tendon and ligament elasticity, decreased joint elasticity, elastic fibrosis due to photoaging, dermal epidermal atrophy, dermal atrophic skin disease, burns, radiation burns, skin lesions, bed sores, dermal aplasia caused by drug administration, or muscle and joint lesions.

11. A functional cosmetic composition for promoting biosynthesis of elastin or collagen comprising the composition according to claim 1.

12. A functional cosmetic comprising the functional cosmetic composition according to claim 11.

13. The functional cosmetic according to claim 12, wherein the functional cosmetic is used for improving skin elasticity and removing wrinkles and/or facial lines.

14. The functional cosmetic according to claim 12, wherein the functional cosmetic is provided in a form of a cream including a facial cream, a hand cream, a moisturizing cream or a sun protection cream, a solution or concentrated solution including a lotion, a cream powder, a foundation, a microemulsion, an ointment, a paste, a pack or a spray.

15. A health functional food beneficial for improving skin health, blood circulation, blood pressure control, or musculoskeletal system health, including joints, comprising the composition according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0020] FIG. 1 shows the results of confirming the ability of the composition according to the present invention to grow fibroblasts (Hs27 cells).

[0021] FIG. 2 shows the results of qPCR confirming the change in the expression levels of COL1A2, ELN and COL4A1 genes in fibroblasts upon treatment with the composition according to the present invention.

[0022] FIG. 3 shows the results of Western blot confirming the production performance of type I collagen and elastin proteins in fibroblasts upon treatment with the composition according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.

[0024] In order to enhance the biosynthesis of collagen and elastin, which are components of dermal tissue, it is known that, when certain amino acids and oligopeptides are properly transported and applied locally or orally, gene expression causing the biosynthesis of dermal and epidermal connective tissues, especially collagen and tropoelastin, is promoted. However, research on an optimal amino acid combination that can promote the production of collagen and elastin in fibroblasts and a mineral composition supporting the same is insufficient. In the present invention, it was found that a composition containing glycine. L-proline and a divalent metal ion enhances the expression of genes involved in biosynthesis of collagen and elastin in fibroblasts, and promotes the production of collagen proteins and elastin proteins in fibroblasts.

[0025] In one aspect, the present invention is directed to a composition for promoting biosynthesis of elastin or collagen containing glycine. L-proline and a divalent metal ion, wherein a content ratio of glycine to L-proline is 1:0.9 to 1:1.1.

[0026] In the present invention, the content ratio of glycine to L-proline is preferably 1:0.9 to 1:1.1, more preferably 1:0.95 to 1:1.05, and most preferably 1:0.99 to 1:1.01.

[0027] The composition of the present invention may further contain one or more amino acids selected from the group consisting of L-alanine, L-valine. L-leucine and L-lysine hydrochloride, and the content ratio of each amino acid to glycine based on weight is preferably as follows: [0028] L-alanine: 0.40 to 0.90, [0029] L-valine: 0.30 to 0.80. [0030] L-leucine: 0.10 to 0.30. [0031] L-lysine hydrochloride: 0.10 to 0.25; [0032] more preferably, [0033] L-alanine: 0.60 to 0.85, [0034] L-valine: 0.40 to 0.75, [0035] L-leucine: 0.15 to 0.25, [0036] L-lysine hydrochloride: 0.12 to 0.20; and

[0037] most preferably, [0038] L-alanine: 0.70 to 0.80, [0039] L-valine: 0.55 to 0.70, [0040] L-leucine: 0.18 to 0.23. [0041] L-lysine hydrochloride: 0.14 to 0.18.

[0042] The composition of the present invention may further contain 0.1 to 30.0% by weight, preferably 0.5 to 25.0% by weight, more preferably 1.0 to 20% by weight of L-cysteine or N-acetyl-L-cysteine based on the total amino acid composition.

[0043] The composition according to the present invention may have a total amino acid content of 1 to 700 g/L, preferably 5 to 650 g/L, more preferably 10 to 600 g/L, and most preferably 15 to 500 g/L, but is not limited thereto.

[0044] In the present invention, the divalent metal may be selected from the group consisting of copper (Cu), cobalt (Co), calcium (Ca), magnesium (Mg), manganese (Mn) and zinc (Zn). The divalent metal ion may be present in an amount of 0.001 to 5.0% by weight, preferably 0.003 to 4.5% by weight, and most preferably 0.005 to 4.0% by weight, based on the metal ion, with respect to the total composition, but is not limited thereto.

[0045] Preferably, the divalent metal ion present in the composition according to the present invention is copper. Copper may be present as a copper metal itself, but is preferably present in the form of a copper salt compound or a hydrate thereof, more preferably in the form of a copper sulfate hydrate, and most preferably in the form of copper sulfate pentahydrate, but is not limited thereto.

[0046] Copper (Cu) used in the composition of the present invention is an essential micro mineral present as a part of various proteins including enzymes in the body while conversion between two valence elements (Cu.sup.+ and Cu.sup.2+) takes place, like iron. Copper is involved in the production of ATP in the final process of the electron transport system in the mitochondria, and copper is bound to superoxide dismutase (SOD) and thus has an antioxidant function of preventing oxidative damage to cells and additionally functions to deposit a melanin pigment in the skin along with manganese and tyrosine (amino acids).

[0047] In one embodiment of the present invention, when treating fibroblasts (Hs27 cells) with the composition containing glycine, L-proline and copper, and detecting changes in the expression levels of the COL1A2 gene, which is a gene involved in type I collagen biosynthesis, an ELN gene, which is a gene involved in elastin biosynthesis, and the COL4A1 gene through qPCR, it was found that the expression of the COL1A2 gene and the ELN gene was increased (see FIG. 2).

[0048] In another embodiment of the present invention, fibroblasts (Hs27 cells) were treated with the composition containing glycine, L-proline and copper, and changes in the production of type I collagen and elastin were detected by Western blot. As a result, it was found that the production ability of the type I collagen and elastin protein increased (see FIG. 3).

[0049] In another aspect, the present invention is directed to a pharmaceutical composition for promoting the biosynthesis of elastin or collagen including the composition.

[0050] The pharmaceutical composition of the present invention can be used for the prevention or treatment of elastin-related diseases, hypertension caused by decreased vascular elasticity and decreased blood circulation, decreased tendon and ligament elasticity, decreased joint elasticity, elastic fibrosis due to photoaging, epidermal atrophy, dermal atrophic skin disease, burns, radiation burns, skin lesions, bed sores, dermal aplasia caused by drug administration, or muscle and joint lesions, but is not limited thereto.

[0051] In addition, the pharmaceutical composition of the present invention can be used for improvement of blood vessel elasticity by induction of elastin and/or collagen and treatment of diseases or amelioration in health conditions through the same.

[0052] The pharmaceutical composition of the present invention may be formulated as an oral administration, an intramuscular injection, a percutaneous injection, a medicine for external application, or a medical device for injection or external application.

[0053] The pharmaceutical composition according to the present invention also contains hyaluronic acid or a salt thereof, particularly hyaluronic acid or a salt thereof, preferably a hyaluronic acid sodium salt, having an average molecular weight of 500,000 to 3,000,000 Da in an amount of 0.01 to 3% by weight of the total composition.

[0054] The composition containing a mixture of the above-described amino acid and hyaluronic acid or a salt thereof is suitable for use in the form of oral administration, intramuscular injection, percutaneous injection, external medicine or a medical device, and may be formulated into a form appropriate therefor and used.

[0055] Non-limiting examples of formulations that can be used include injections, gels, ointments, emulsions, transdermal patches, sterile solutions, fillers, wound coatings and amino acid powders designed to be reconstituted with a sterile aqueous solution of hyaluronic acid or a salt thereof.

[0056] Formulations for injection are prepared by dissolving an amino acid in the form of a powder in a sterile solution of hyaluronic acid or a salt thereof, preferably by directly introducing the hyaluronic acid or a salt thereof, preferably, a sodium hyaluronate gel, into a vial containing a powder (e.g., using a skin graft syringe). After complete dissolution, the resulting gel solution is injected into the dermal site. The sterile aqueous solution of hyaluronic acid or a salt thereof may contain a pH-adjusting buffer (e.g. phosphate buffer) or an osmotic pressure adjusting agent (e.g. sodium chloride), and other technical adjuvants, which can ensure the physicochemical and tissue compatibility required for sterile injectable pharmaceutical formulations.

[0057] In another aspect, the present invention is directed to a cosmetic composition, in particular a functional cosmetic composition including the composition for promoting the biosynthesis of elastin or collagen according to the present invention.

[0058] The functional cosmetic composition according to the present invention may be used for improving skin elasticity and removing wrinkles and/or facial lines, but is not limited thereto.

[0059] Depending on the purpose of use and functions thereof, the cosmetic composition of the present invention can be formulated in various forms such as a solution (lotion-type composition), concentrated solution, gel, ointment, emulsion (cream, emulsion), vesicle dispersion, powder, dense powder, paste or solid, and can be provided as a functional cosmetic.

[0060] Specifically, the cosmetic containing the cosmetic composition according to the present invention includes all products including creams such as facial creams, hand creams, moisturizing creams and sun protection creams, solutions or concentrated solutions, lotions, cream powders, foundations, microemulsions, ointments, pastes, packs and sprays, but is not limited thereto.

[0061] When the composition of the present invention is used for improvement of elasticity and removal of wrinkles and facial lines, and as a moisturizing agent, the compositions for these applications are preferably provided in the form of emulsions such as creams, gels, lotions, ointments, or vesicle dispersions, and may further include other pharmaceutical or functional active ingredients.

[0062] The functional cosmetic composition may contain modified or unmodified oils of vegetable or animal oils. Examples of these oils include sweet almond oil, avocado oil, castor oil, olive oil, jojoba oil, sunflower oil, wheat flour oil, sesame oil, peanut oil, grapeseed oil, soy milk, safflower oil, coconut oil, maize oil, hazelnut oil, Karite butter, palm oil, apricot kernel oil, calophyllum oil, or perhydrosqualene. Moreover, the oil phase may be an inorganic oil such as liquid paraffin or liquid petrolatum. The oil may be a fatty acid ester such as isopropyl myristate, isopropyl palmitate, 2-ethylhexyl palmitate, penicillin oil (stearyl octonate), unsaturated fatty acid such as oleic acid, palmitic acid, stearic acid, behenic acid, linoleic acid and linolenic acid, or a volatile or nonvolatile isoparaffin such as fatty acid or C8-C16 isoparaffin. The oil may be a C12-C18 fatty alcohol such as oleyl alcohol, cetyl alcohol or stearyl alcohol.

[0063] When the composition is provided as an emulsion, the emulsified composition of the present invention contains an oil phase and an aqueous phase. The oil phase is preferably provided in the range of about 1 to about 75% by weight, more preferably about 5 to about 60% by weight, and most preferably about 40 to about 60% by weight, based on the total weight of the composition.

[0064] The aqueous phase contains an adjuvant commonly used in aqueous gels and cosmetic emulsions. The aqueous phase may be provided in an amount from about 0.5 to about 20% by weight, and may include lower C2-C6 mono-alcohols and/or polyols such as glycerol, butylene glycol, isoprene glycol, propylene glycol, ethylene glycol and the like.

[0065] The emulsifier can be used for the preparation of the emulsified cosmetic composition. A cosmetically acceptable emulsifier can be used in any amount as long as it exhibits the desired emulsion effect. The emulsifier is selected from known salts and surfactants. The emulsifier is preferably selected from stearic acid, sorbitan sesquioleate, polyethylene glycol (PEG-30), dipolyhydroxystearate, lecithin, magnesium stearate, and derivatives and mixtures thereof. The emulsifier is preferably provided in a range of about 0.5 to about 30% by weight based on the total weight of the composition, more preferably about 1 to about 12% by weight, and more preferably about 4 to about 8% by weight.

[0066] In another aspect, the present invention is directed to a health functional food containing the composition for promoting biosynthesis of elastin or collagen according to the present invention as an active ingredient.

[0067] The functional food according to the present invention can be used for skin health, improvement in blood circulation, blood pressure control, or prevention and treatment of musculoskeletal diseases including joint diseases, specifically improvement in skin elasticity, removal of wrinkles, removal of facial lines, alleviation of hypertension, improvement in blood circulation, improvement of elasticity of tendons and ligaments, improvement of joint elasticity, and prevention or treatment of elastic fibrosis due to photoaging, dermal epidermal atrophy, dermal atrophic skin disease, burns, radiation burns, skin lesions, bed sores, and dermal aplasia or muscle and joint lesions caused by drug administration, and is preferably used for cosmetic application or joint ailment prevention.

[0068] In addition, the functional food of the present invention can be utilized in various applications including drugs, foods and beverages for preventing oxidation. The functional food of the present invention may be used as, for example, various foods, candy, chocolate, beverages, gum, tea, vitamin complexes, health supplements and the like, and may be used in the form of powders, granules, tablets, capsules or beverages.

[0069] The composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, and organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These ingredients may be used independently or in combination.

[0070] In another aspect, the present invention is directed to a method for promoting biosynthesis of elastin or collagen including administering the composition.

[0071] In another aspect, the present invention is directed to the use of the composition for enhancement of elastin or collagen biosynthesis.

[0072] In another aspect, the present invention is directed to the use of the composition for the preparation of a drug for promoting elastin or collagen biosynthesis.

EXAMPLE

[0073] Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.

Example 1: Confirmation of Toxicity to Fibroblasts of Composition According to Present Invention

[0074] The composition according to the present invention was prepared as shown in Table 1 below, and the cytotoxicity to fibroblasts of the composition of Table 1 was determined.

[0075] The fibroblasts herein used were human-derived fibroblasts Hs27 (ATCC, #CRL 1634) and the cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin.

[0076] A drug prepared in a dose of 1000 μg/10 μl was diluted in serum-free DMEM medium containing 1% penicillin-streptomycin for treatment of cells.

TABLE-US-00001 TABLE 1 Content of each ingredient in formulation (μg/10 μl) Ingredient D EL1 EL4 EL11 EL12 Glycine 302 266.7 266.7 266.7 266.7 L-proline 227 266.7 266.7 266.7 266.7 L-alanine 228 200 200 200 200 L-valine 168 166.7 166.7 166.7 166.7 L-leucine 42 56.7 56.7 56.7 56.7 L-lysine HCl 33 43.3 43.3 43.3 43.3 Sum 1000 1000.1 1000.1 1000.1 1000.1 Copper sulfate 0 0 3.3 1.67 0.33 pentahydrate Total 1000 1000.1 1003.4 1001.77 1000.43

[0077] 1×10.sup.5 Hs27 cells were seeded in a 48-well plate and cultured for 24 hours. After 24 hours, the cells were treated with the drug prepared above. In order to observe cytotoxicity, an EZ-cytox (Dogen Bio, Korea) kit for measuring cell viability was used after 24 hours, 48 hours and 72 hours. Water soluble tetrazolium salts (WST) that reacted with enzymes present in viable cells contained in KIT was treated, absorbance at 450 nm was measured, and cell viability was calculated.

[0078] As a result, as shown in FIG. 1, compared to the NC group treated only with the solvent, it was found that the cells treated with d, EL1, EL11 and EL12 did not show cytotoxicity even after 24 hours, 48 hours or 72 hours. It was found that the cells treated with EL4 did not show toxicity after 24 hours or 48 hours, but the cell viability thereof decreased slightly after 72 hours.

Example 2: Determination of COL1A2, COL4A1 and ELN Gene Expression Levels Upon Treatment with Composition According to Present Invention

[0079] Changes in the expression levels of genes involved in the biosynthesis of collagen and elastin in Hs27 cells according to the treatment of the composition according to the present invention were determined.

[0080] 5×10.sup.6 Hs27 cells were seeded in a 100π dish and cultured for 24 hours. The cells were treated with the prepared compositions (d, EL1, EL11 and EL12) after 24 hours. RNA was extracted using TRIzol after 72 hours to determine the gene expression level. 1 μg of RNA extracted from each sample was synthesized into cDNA using a cDNA reverse transcriptase, and the expression levels of COL1A2, ELN, COL4A1 and ACTB genes were detected using a real-time PCR detection system (Biorad CFX Connect). In order to identify each gene, the primers of Table 2 were used, and relative expression levels were calculated using ACTB (β-actin) as an internal control.

TABLE-US-00002 TABLE 2 Primer sequences for detecting gene expression level Gene Forward Reverse COL1A2 CCA AAT CTG TCT CCC TCA AAA ACG AAG GGG CAG AA (SEQ ID NO: 1) AGA TG (SEQ ID NO: 2) ELN CCA TGT CCA CAC AAG GCC AGA GTG GCT TTC GAC AG (SEQ ID NO: 3) TCA AC (SEQ ID NO: 4) COL4A1 GCT TGA AAA GGG TTG TTG AGT CCC GGT AGA AGC AG (SEQ ID NO: 5) CCA AC (SEQ ID NO: 6) ACTB GAG GCC TGG ACT CTC AAT GAA TGG GGG TTG AAC TG (SEQ ID NO: 7) AAT GA (SEQ ID NO: 8)

[0081] As a result, as shown in FIG. 2, compared to the NC group treated only with solvent, it was found that there was no significant change in the expression levels of the COL1A2 gene and the ELN gene in the cells treated with the composition d, EL1 and EL12 after 72 hours, and it was found that the expression levels of the COL1A2 gene and ELN gene in the cell groups treated with EL4 and EL11 were significantly increased after 72 hours. In addition, unlike the COL1A2 gene and the ELN gene, it was found that there was no change in the expression level in the COL4A1 gene, under all composition treatment conditions.

Example 3: Confirmation of Protein Production Ability of COL1A2, COL4A1, ELN Upon Treatment with Composition According to Present Invention

[0082] Changes in the ability to produce collagen and elastin proteins in Hs27 cells upon treatment with the composition according to the present invention were detected.

[0083] 5×10.sup.6 Hs27 cells were seeded in a 100π dish and cultured for 24 hours. The cells were treated with the prepared compositions (d, EL1, EL11 and EL12) after 24 hours. To detect the protein expression level, after 72 hours, the protein was extracted using a protein lysis buffer (cell signaling, #9803). Western blotting was performed on each protein by the following method to identify type I collagen, elastin and D-actin proteins.

[0084] Type I collagen: Western blot was performed in a native environment (non-denaturing condition) to determine the protein expression level of type I collagen. A sample was prepared by diluting 30 μg of the extracted protein in a native sample buffer (Biorad, #1610738).

[0085] Electrophoresis was performed using an 8% acrylamide gel containing no SDS and a tank buffer (Tris-glycine buffer), and the protein separated on the gel was transferred to the PVDF membrane and then blocked with skim milk for 2 hours. After that, the protein was allowed to stand at 4° C. overnight using an anti-collagen I antibody (abcam, ab34710) and was then reacted with a secondary antibody for 1 hour. The reaction of the antibody was detected using an ECL solution and an Amersham Imager 600 imaging system.

[0086] Elastin and β-actin: Western blot was performed in a general Western blot environment (non-native and denaturing conditions) to detect the protein expression levels of elastin and β-actin. A sample was prepared by diluting 30 μg of the extracted protein in a 4× Laemmli sample buffer (Biorad, #1610747). Electrophoresis was performed using 10% acrylamide gel containing SDS and a tank buffer (Tris-glycine buffer). Then, the protein separated on the gel was transferred to the PVDF membrane and blocked using skim milk for 2 hours. Then, the protein was allowed to stand at 4° C. overnight using Anti-Elastin antibody (Santacruz, sc-166543) and Anti-β-actin antibody (Santacruz, sc-47778) and then reacted with a secondary antibody for 1 hour. The reaction of the antibody was detected using an ECL solution and an Amersham Imager 600 imaging system.

[0087] As a result, as shown in FIG. 3, compared to the NC group treated with a solvent, the cells treated with the d, EL1 and EL12 compositions were found to show an increase in the protein expression level of type I collagen after 72 hours. Elastin was not changed in the groups treated with d and EL1, but was significantly increased in the groups treated with the EL4, EL11 and EL12 compositions.

INDUSTRIAL AVAILABILITY

[0088] The composition according to the present invention can enhance the biosynthesis of elastin and collagen of fibroblasts and is thus useful for restoring skin elasticity deteriorated due to aging or exposure to light and for removing wrinkles and facial lines.

[0089] Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this description is provided to set forth preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalents thereto.