PRODUCTION PROMOTER OF TYPE I COLLAGEN OR ELASTIN

Abstract

A type I collagen or elastin production promoter containing a mycosporine-like amino acid or a salt thereof as an active ingredient; and a method for promoting type I collagen or elastin production, the method including administering an effective amount of a mycosporine-like amino acid or a salt thereof.

Claims

1-12. (canceled)

13. A method for promoting type I collagen or elastin production, the method comprising administering an effective amount of a mycosporine-like amino acid or a salt thereof.

14. The method according to claim 13, wherein the mycosporine-like amino acid or the salt thereof is derived from algae.

15. A method for promoting type I collagen gene or elastin gene expression, the method comprising administering an effective amount of a mycosporine-like amino acid or a salt thereof.

16. The method according to claim 15, wherein the mycosporine-like amino acid or the salt thereof is derived from algae.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a graph illustrating the effect of mycosporine-like amino acids (MAAs) in promoting elastin gene expression.

[0029] FIG. 2 is a graph illustrating the effect of mycosporine-like amino acids (MAAs) in promoting type I collagen gene expression.

DETAILED DESCRIPTION OF THE INVENTION

[0030] An active ingredient of a type I collagen or elastin production promoter or a type I collagen or elastin gene expression promoter according to the present invention is a mycosporine-like amino acid or a salt thereof.

[0031] Mycosporine-like amino acids are amino acids having a cyclohexanone structure and known to be produced by algae such as seaweed and microalgae, marine products, and fungi, and other organisms. Mycosporine-like amino acids have been found as ultraviolet absorbing substances. Although mycosporine-like amino acids are known to have an effect of absorbing ultraviolet radiation and an effect of promoting fibroblast growth as described above, they are not known to have an effect of promoting type I collagen and elastin production.

[0032] The following compounds are known as examples of mycosporine-like amino acids.

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[0033] Mycosporine-like amino acids or salts thereof used in the present invention can be extracted from, for example, marine products such as shellfish, and fungi, but preferably derived from algae, more preferably derived from blue-green algae, red algae, brown algae, or green algae. Mycosporine-like amino acids derived from algae include the above compounds.

[0034] To extract mycosporine-like amino acids from algae or shellfish, a polar solvent, such as water, ethanol, acetone, methanol, isopropanol, acetonitrile, or a mixed solvent thereof, can be used to extract mycosporine-like amino acids from dried or freeze-dried algae. The extraction solvent is preferably a water-ethanol mixture and more preferably a mixture of water:ethanol=1:9 to 9:1. The obtained extract is preferably concentrated by adsorbing mycosporine-like amino acids to an activated-carbon adsorption column, a resin column, or other columns, and eluting them with a water-ethanol mixture. The mycosporine-like amino acids can be purified by high performance liquid chromatography.

[0035] An algal extract containing mycosporine-like amino acids is already commercially available, and such a commercially available extract can also be used.

[0036] Examples of salts of mycosporine-like amino acids include acid addition salts, such as hydrochlorides, and alkali metal salts, such as sodium salts and potassium salts.

[0037] Mycosporine-like amino acids or salts thereof have a strong effect of promoting type I collagen and elastin production as described below in Examples. Such a preferable effect of promoting type I collagen and elastin production is so strong that it cannot be expected from the fibroblast growth promoting effect of mycosporine-like amino acids.

[0038] Mycosporine-like amino acids or salts thereof have an effect of strongly promoting type I collagen gene or elastin gene expression. The effect of mycosporine-like amino acids in promoting type I collagen or elastin production is thus considered to be based on the effect of promoting the type I collagen gene or elastin gene expression.

[0039] The type I collagen or elastin production promoter according to the present invention has an effect of promoting type I collagen or elastin production in various sites in vivo. The type I collagen or elastin production promoter according to the present invention is thus useful as, for example, pharmaceuticals, quasi-drugs, cosmetics, foods for specified health uses, foods with functional claims, functional foods, beauty foods, and foods for sick people in order to provide, for example, drugs for relieving joint pain or improving joint movement and drugs for improving skin elasticity.

[0040] Examples of the dosage form of the type I collagen or elastin production promoter according to the present invention include oral dosage forms, injection dosage forms, and transdermal dosage forms. For use as, for example, pharmaceuticals, quasi-drugs, or foods with functional claims, the type I collagen or elastin production promoter according to the present invention is preferably in the form of preparations for oral administration, such as tablets, granules, fine granules, capsules, and troches; or skin external preparations, such as creams, ointments, emulsions, and lotions. For use as foods for specified health uses or foods with functional claims, the type I collagen or elastin production promoter according to the present invention may be in the same form as common foods, such as beverages and yogurt.

[0041] The amount of a mycosporine-like amino acid or a salt thereof in such a preparation is preferably, but not necessarily, from 0.001 to 50 mass %, more preferably from 0.001 to 30 mass %.

[0042] The preparations for oral administration can further contain, for example, an excipient, a binder, a disintegrant, a lubricant, a colorant, a corrigent, and a deodorant, in addition to the mycosporine-like amino acid or the salt thereof. The skin external preparations can further contain, for example, various oils, a coloring agent, a preservative, a surfactant, and a thickener, in addition to the mycosporine-like amino acid or the salt thereof. The skin external preparations can further contain a hyaluronic acid production promoter.

EXAMPLES

[0043] Next, the present invention will be more specifically described by way of Examples.

Text Example 1 (Epidermal Cytotoxicity Test)

1) Conditions for Culturing Human Normal Skin-Derived Epidermal Cells

[0044] Human normal skin-derived epidermal cells were cultured by using HuMedia-KG2 (medium for epidermal cells) in a CO.sub.2 incubator (5% CO.sub.2, 37° C.). The medium was exchanged every other day.

2) Cytotoxicity Test

[0045] Normal human epidermal cells were seeded in a 96-well plate at a concentration of 2×10.sup.4 cells/100 μL/well. The next day, the medium was replaced with media (100 μL/well) having different sample concentrations (10-fold dilution series with 10% being the highest concentration, 8 levels of concentration), followed by 24-hour culturing. The cell morphology was observed under a phase-contrast microscope at the end of culturing, and the medium was removed, followed by washing with PBS once. A medium (100 μL) supplemented with 10% of viable cell count reagent SF was then added, followed by incubation at 37° C. in 5% CO.sub.2 for 90 minutes. During incubation, the absorbance of the sample was measured by using a microplate reader every 30 minutes (measurement wavelength: 450 nm, control wavelength: 595 nm). The cell viability was calculated as O.D. values after 60 minutes to 0 minutes. The cell viability was expressed as a relative value when the cell viability in the negative control test section was defined as 100%. The test was carried out at n=3 for each test concentration.

3) Results

[0046] No cytotoxicity was observed even after the cells were cultured in the presence of 0.00001 mg/mL to 10 mg/mL of mycosporine-like amino acids (dried product of mycosporine-like amino acids extracted and fractionated with a 70% ethanol aqueous solution and an activated carbon column from algae, as in JP 2007-16004 A) serving as samples.

Test Example 2 (Fibroblast Growth Test)

1) Culture Conditions

[0047] Normal human fibroblasts were cultured by using a 10% FBS-supplemented D-MEM in a CO.sub.2 incubator (5% CO.sub.2, 37° C.) The 10% FBS-supplemented D-MEM was prepared by adding 50 mL of FBS and 5 mL of penicillin-streptomycin solution to 445 mL of D-MEM.

2) Cell Activation Test (WST-8 Assay)

[0048] Fibroblasts prepared at 5×10.sup.4 cells/mL in a growth medium were seeded at 100 μL per well (96-well plate) (5×10.sup.3 cells/100 μL/well). This is the number of cells to be approximately 50% confluent and in the logarithmic growth phase the next day, which is suitable for analyzing the effect of a test substance on the cell growth activation. The fibroblasts were cultured for about 24 hours after seeding, and the growth medium was replaced with 100 μL of growth media having different sample concentrations, followed by 24-hour culturing. The media were removed at the end of culturing, and 100 μL of a medium supplemented with 10% of viable cell count reagent SF was added, followed by incubation at 37° C. in 5% CO.sub.2 (measurement wavelength: 450 nm, control wavelength: 595 nm). After addition, the absorbance was measured at 30 minutes and 90 minutes by using a microplate reader (measurement: 450 nm), and the change in absorbance per hour was calculated from the absorbances at 30 minutes and 90 minutes. The test was carried out at n=5 for each test concentration.

3) Results

[0049] The relative viable cell count in the group with addition of 1 mg/mL of mycosporine-like amino acids was 109±2.22%, which was about 10% higher than 100±1.43% in the negative control group and higher than that in the negative control group with a significance (p<0.05).

TABLE-US-00001 TABLE 1 Cell Viability(%) Relative Negative MAAs(mg/mL) value (%) control 0.0016 0.008 0.04 0.2 1 10% FBS 101 89 86 86 96 105 122 102 86 91 90 97 104 108 102 86 89 96 94 111 100 100 81 87 85 102 116 99 94 86 74 77 72 107 100 Mean 100 86 86 87 94 109 106 Standard 1.43 1.22 2.91 3.05 3.94 2.22 4.36 deviation

Test Example 3 (Gene Expression Test)

[0050] The medium used was D-MEM (Sigma-Aldrich)+10% FBS (Biowest), and human normal fibroblasts (HDFn, Gibco) were seeded at 9.0×10.sup.4/well (12-well plate). After colonization, the medium was replaced with a starvation medium (D-MEM (Sigma-Aldrich)) for 24 hours. Subsequently, the medium was replaced with D-MEM prepared so as to contain 0, 10, 25, 30, 40, and 50 μg/mL of mycosporine-like amino acids. The cells were collected after 3 hours, and the RNA was extracted by using a RNA extraction kit (ReliaPrep™ RNA Miniprep Systems, catalog no. Z6012, Promega Corporation). The cDNA was then synthesized (Toyobo Co., Ltd., product number FSQ-301S), and the type I collagen gene or elastin gene expression was analyzed by real-time RT-PCR (SYBR Fast qPCR Mix, catalog no. RR430B, Takara Bio Inc.).

[0051] The following primers were used. To normalize the gene expression, the gene expression level was corrected with the expression level of the Ribosomal Protein Lateral Stalk Subunit P0 (RPLPO) gene.

TABLE-US-00002 TABLE 2 Primer Sequence Elastin Forward 5′-AGGTGTATACCCAGGTGGCGTGCT-3′ (SEQ ID NO: 1) Reverse 5′-CAACCCCTGTCCCTGTTGGGTAAC-3′ (SEQ ID NO: 2) Type I Forward 5′-CTGGTCCCCAAGGCTTCCAAGGTC-3′ (SEQ ID NO: 3) collagen Reverse 5′-CCATCATTTCCACGAGCACCAGCA-3′ (SEQ ID NO: 4) RPLP0 Forward 5′-TTCGACAATGGCAGCATCTACAA-3′ (SEQ ID NO: 5) Reverse 5′-CTGCAGACAGACACTGGCAACA-3′ (SEQ ID NO: 6)

[0052] As a result, the elastin gene expression and the type I collagen gene expression were both significantly high at 30 μg/mL of mycosporine-like amino acids as shown in FIG. 1 and FIG. 2.