JP2008285472A - Extracellular matrix splitting enzyme inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new application of an organic acid as an extracellular matrix splitting enzyme inhibitor. <P>SOLUTION: It has been found that organic acids including pyroglutamic acid, succinic acid, malic acid, lactic acid and acetic acid function as an extracellular matrix splitting enzyme inhibitor each. The extracellular matrix splitting enzyme inhibitor functions particularly effectively as a gelatinase A inhibitor. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an extracellular matrix degrading enzyme inhibitor.

  Organic acids such as pyroglutamic acid, citric acid, succinic acid, malic acid, lactic acid, acetic acid and tartaric acid are contained in natural foods and the like, and are widely used industrially in the food and chemical fields.

  Pyroglutamic acid is a kind of amino acid and is also called PCA (Pyrrolidene Carboxylic Acid). In vivo, free glutamic acid is affected by temperature and pH, whereby the amino group and the carboxyl group of the side chain are bonded and cyclized to produce pyroglutamic acid. Pyroglutamic acid is widely contained in food materials such as vegetables, fruits, dairy products, and meat, and is also present in large amounts in human brain, spinal fluid, and blood. Conventionally, pyroglutamic acid is known to be involved in the activity and production of neurotransmitters and stimulate the brain to enhance memory (see Patent Document 1).

Citric acid, succinic acid, lactic acid, tartaric acid and the like are used as food additives in the brewing industry, beverages and food production. Malic acid is used as an acidulant in soft drinks and processed foods, and as an emulsifying stabilizer, deodorant, detergent, and neutralizing agent for industrial use. Acetic acid is widely used in photography, reagents, dyeing, food, medicine and the like (Non-patent Document 1).
Japanese Patent Laid-Open No. 5-117232 15107 chemical products, January 2007, Chemical Daily

  However, organic acids that are contained in many natural food materials and are highly safe and easily available are expected to find further useful applications.

  That is, an object of the present invention is to provide a new use of an organic acid.

  As a result of studies on further useful uses of the organic acid, the present inventors have found that the organic acid has an inhibitory activity on extracellular matrix degrading enzyme activity, and have completed the present invention. That is, the present invention provides an extracellular matrix degrading enzyme inhibitor comprising an organic acid.

  The organic acid preferably contains at least one selected from the group consisting of pyroglutamic acid, citric acid, succinic acid, malic acid, lactic acid, tartaric acid, malonic acid, acetic acid, phenylalanine, tryptophan, and tyrosine. By using these organic acids, an extracellular matrix degrading enzyme inhibitor having excellent inhibitory activity can be provided.

  The extracellular matrix degrading enzyme inhibitor of the present invention exhibits an excellent anti-wrinkle effect particularly by having gelatinase A inhibitory activity.

  ADVANTAGE OF THE INVENTION According to this invention, the new use as an extracellular-matrix degradation enzyme inhibitor of an organic acid can be provided.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  The extracellular matrix degrading enzyme inhibitor of the present invention comprises an organic acid.

  Among the organic acids in the present invention, pyroglutamic acid, malic acid, tartaric acid, lactic acid, phenylalanine, tryptophan and tyrosine include their stereoisomers and tautomers. That is, pyroglutamic acid, malic acid, tartaric acid, lactic acid, phenylalanine, tryptophan, and tyrosine have two stereoisomers (D and L), all of which function as extracellular matrix degrading enzyme inhibitors. Accordingly, the extracellular matrix degrading enzyme inhibitor of the present invention may consist of only one stereoisomer or a mixture of two stereoisomers in any ratio (for example, a racemate) Is good).

  D-pyroglutamic acid is also known by a name such as D-pyrrolidonecarboxylic acid, and L-pyroglutamic acid is also known by a name such as L-pyrrolidonecarboxylic acid.

  An extracellular matrix degrading enzyme inhibitor is a substance having an inhibitory action on extracellular matrix degrading enzyme activity. The extracellular matrix-degrading enzyme is an enzyme involved in extracellular matrix metabolism, also called matrix metalloproteinase, matrix metalloproteinase (MMP), or the like.

  In general, connective tissues of animals are composed of an extracellular matrix mainly composed of collagen or proteoglycan. The metabolism of extracellular matrix is regulated by the balance between extracellular matrix degrading enzyme (hereinafter referred to as “MMP”) and in vivo inhibitory factor (TIMP: Tissue Inhibitor of Metalloproteinase). Among these, MMP is It is believed that degradation or denaturation of the extracellular matrix causes a decrease in skin elasticity and the generation of wrinkles and talmi associated therewith.

  Among the MMPs, MMP1 (collagenase) uses collagen I, which is a collagen fiber distributed in the dermis, as a substrate. Distributing V-type collagen, elastin, which is an elastic fiber, fibronectin, which is a glycoprotein distributed in soft connective tissue and basement membrane, and the like are used as substrates.

  Thus, MMP1 and MMP2 are considered to be particularly related to the generation of wrinkles and talmi in the skin because they decompose or denature important substrates for maintaining skin firmness. Moreover, since MMP1 and MMP2 are activated when ultraviolet rays are applied to the skin, it is considered that MMP1 and MMP2 are also involved in damage to the skin matrix by ultraviolet rays.

  The MMP inhibitor of the present invention inhibits the activity of MMP2 (gelatinase A) among these MMPs, thereby preventing the occurrence of skin wrinkles and talmi, and exerting the effect of keeping the skin firm. That is, the MMP inhibitor of the present invention functions as a skin wrinkle inhibitor, a skin talmi inhibitor, a skin tension improving agent, and the like.

  The MMP inhibitor of the present invention can be used as a skin external preparation. In that case, it can be applied directly to the skin, or other substances may be mixed and used.

  The MMP inhibitor of the present invention can be used in cosmetics, toiletries, oral care products, foods, beverages or pharmaceuticals. Cosmetics, foods, beverages or pharmaceuticals containing the MMP inhibitor of the present invention include an organic acid as an active ingredient, a wetting agent, an oily component, a moisturizer, a powder, a pigment, an emulsifier, a dispersion aid, a solubilizer, It may contain a cleaning agent, an ultraviolet absorber, a thickener, a drug, a fragrance, a resin, an excipient, an antibacterial and antifungal agent, a deodorant / deodorant, an enzyme, purified water, and alcohol. Further, other MMP inhibitors may be added.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. Further, unless otherwise specified, “%” represents “mass%”.

Example 1
MMP2 inhibitory activity of organic acids Lactic acid, acetic acid, formic acid, propionic acid, pyruvic acid, fumaric acid, maleic acid, malonic acid, tartaric acid, malic acid, succinic acid, citric acid and pyroglutamic acid were used as samples.

In a reaction vessel, 58 mU / μL of E. coli. E. coli recombinant human (E. coli recombinant human) MMP2 enzyme solution and a solution of the sample dissolved in buffer (50 mM HEPES, 10 mM CaCl ₂ , 0.05% Brij-35, 1 mM DTNB, pH 7.5), A reaction solution containing a 1% concentration sample was prepared. After incubation at 37 ° C. for 60 minutes, 1 mM Thiopeptide solution (main component is oligoprotein Ac-PLG- [2-mercapto-4-methyl-pentanoyl] -LG-OC ₂ H ₅ ) as an extracellular matrix simulated substrate solution. Added to the reaction. A thiol produced by the cleavage of the disulfide bond of DTNB (5,5'-dithiobis (2-nitrobenzoic acid) by the sulfurhydryl (sulfhydryl) group generated by cleaving Thiopeptide by MMP2 was detected at an absorbance of 412 nm. What added the buffer instead of the solution was used as control.

Absorbance at 412 nm was measured for 60 minutes from the time when the simulated extracellular matrix solution was added after incubation. The increase in absorbance over time for 60 minutes was approximated by a linear equation, and the slope was defined as the amount of change in absorbance. The inhibition rate was calculated | required by the following formula using the obtained change amount of absorbance. In the following formulas, ΔABS _sample and ΔABS _control represent the amount of change in absorbance obtained for the sample (reaction solution) and the control, respectively.
Inhibition rate (%) = [(ΔABS _control− ΔABS _sample ) / ΔABS _control ] × 100

Table 1 shows the MMP2 inhibitory activity at 1% concentration of each sample in terms of the inhibition rate (%).

  As shown in Table 1, MMP2 inhibitory activity in a 1% solution of each organic acid was confirmed. Among them, MMP2 inhibitory activity in a 1% solution of pyroglutamic acid, maleic acid, malic acid, malonic acid, succinic acid, tartaric acid and citric acid was higher than that of other organic acids.

(Example 2)
Further, among the organic acids having high inhibitory activity, MMP2 activity at various concentrations was measured for pyroglutamic acid, citric acid, malic acid and succinic acid.

Table 2 shows the concentration of each sample when the MMP2 activity is inhibited by 50% (IC50 value: 50% inhibitory concentration).

  As shown in Table 2, it was confirmed that pyroglutamic acid, citric acid, malic acid and succinic acid have very low IC50 values and are useful as MMP2 inhibitors.

(Example 3)
As a sample, L-phenylalanine, L-tryptophan, and L-tyrosine were used.

In a reaction vessel, 58 mU / μL of E. coli. E. coli recombinant human (E. coli recombinant human) MMP2 enzyme solution and a solution prepared by dissolving the sample in a buffer solution (50 mM HEPES, 10 mM CaCl ₂ , 0.05% Brij-35, pH 7.5) were added. A reaction solution containing a sample with the indicated concentration was prepared. After incubation at 37 ° C. for 30 minutes, 0.4 mM “OmniMMP fluorogenic substrate peptide” (trade name, manufactured by BIOMOL) was added to the reaction solution as an extracellular matrix simulated substrate solution. From the substrate cleaved by MMP2, 380 nm fluorescence generated by 340 nm excitation light was detected. What added the buffer instead of the sample buffer solution was used as control.

The fluorescence intensity at 380 nm was measured for 20 minutes from the time when the extracellular matrix simulated substrate solution was added after the incubation. The increase in fluorescence intensity over time for 20 minutes was approximated by a linear expression, and the slope was defined as the amount of change in fluorescence intensity. On the other hand, a calibration curve is prepared using a solution in which the standard fluorescent reagent “OmniMMP fluorogenic control peptide” (manufactured by BIOMOL, trade name) is added in a concentration step to the reaction solution or control to which the substrate solution is not added, The slope was used as a conversion coefficient. Using the obtained fluorescence intensity change amount and conversion coefficient, the inhibition rate was determined by the following formula. In the following formulas, ΔABU _sample and ΔABU _control represent the amount of change in fluorescence intensity obtained for the sample (reaction solution) and the control, respectively. CF _sample and CF _control represent the conversion coefficients obtained for the sample (reaction solution) and the control, respectively.
Inhibition rate (%) = [((ΔABU _control / CF _control ) − (ΔABU _sample / CF _sample )) / (ΔABU _control / CF _control )] × 100

Table 3 shows the MMP2 inhibitory activity of each sample at the concentration (%) shown in the table in terms of the inhibition rate (%).

  As shown in Table 3, phenylalanine and tryptophan have very low IC50 values of 0.24% and 0.16%, respectively, and tyrosine has an MMP2 as high as 41% at a very low concentration of 0.04%. It was confirmed to have inhibitory activity. That is, it was confirmed that phenylalanine, tryptophan and tyrosine are useful as MMP2 inhibitors.

  The MMP inhibitor provided by the present invention is suitably used as cosmetics, toiletries, oral care products, foods, beverages or pharmaceuticals.

Claims (3)

  An extracellular matrix degrading enzyme inhibitor comprising an organic acid.   2. The organic acid according to claim 1, wherein the organic acid contains at least one selected from the group consisting of pyroglutamic acid, citric acid, succinic acid, malic acid, lactic acid, tartaric acid, malonic acid, acetic acid, phenylalanine, tryptophan, and tyrosine. Extracellular matrix degrading enzyme inhibitor.   The extracellular matrix degrading enzyme inhibitor according to claim 1 or 2, which is a gelatinase A inhibitor.