Hyaluronidase activity inhibitor

Abstract

To provide a hyaluronidase inhibitor with high pharmacological activity, which inhibits hyaluronidase activity that decomposes hyaluronic acid, besides a method of compensating a decreased amount by direct oral ingestion of the hyaluronic acid.SOLUTION: A hyaluronidase activity inhibitor is characterized by an active ingredient α- ketoglutaric acid. A concentration of the α- ketoglutaric acid is 0.2 to 4 mM. Accordingly, a decrease in the hyaluronic acid can be suppressed, and an amount of the hyaluronic acid can be maintained.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting hyaluronidase activity. [Background technology]

[0002] In recent years, research into preventing skin aging has been widely conducted. While aging is an important factor in causing skin aging, the effects of dryness, oxidation, and ultraviolet rays are also cited as direct factors involved in skin aging. Specific phenomena known to contribute to skin aging include a decrease in mucopolysaccharides such as hyaluronic acid, collagen cross-linking, and cell damage caused by ultraviolet rays.

[0003] Hyaluronic acid is widely present in organs and tissues such as the skin, joints, ligaments, lungs, kidneys, and brain. In particular, in the skin, it is an important factor in maintaining moisture due to its high moisturizing properties (Non-Patent Document 1). It is also known that the amount of hyaluronic acid present in the skin accounts for 50% of the total amount in the body (Non-Patent Document 2).

[0004] To maintain skin moisture, it is important to maintain the amount of hyaluronic acid. Therefore, methods to replenish decreased hyaluronic acid and methods to prevent the decomposition of hyaluronic acid are considered to maintain the amount of hyaluronic acid. Proposed methods for replenishing hyaluronic acid include oral ingestion, in which hyaluronic acid is taken directly through the mouth, and transdermal absorption, in which hyaluronic acid is taken through the skin. It has been reported that orally ingested hyaluronic acid is first broken down into smaller molecules by intestinal bacteria, then absorbed through the intestinal tract and resynthesized in the skin (see Non-Patent Document 3). On the other hand, in the case of transdermal absorption, it is known that hyaluronic acid is not absorbed through the skin due to its large molecular weight. However, the water-retaining properties of hyaluronic acid have been confirmed to prevent skin dryness.

[0005] On the other hand, hyaluronidase can be mentioned as a substance that decomposes hyaluronic acid. Hyaluronidase is known as an enzyme that hydrolyzes hyaluronic acid. Indomethacin, aspirin, etc. are known as substances that inhibit the activity of hyaluronidase (hereinafter referred to as "hyaluronidase activity inhibitors"). In addition, rosmarinic acid has been reported as a naturally occurring hyaluronidase inhibitor (see Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 9-067251 [Non-patent literature]

[0007] [Non-Patent Document 1] Papakonstantinou E.,:Hyaluronic acid : A Keymoleculein skin aging, Dermatoendocrinol, 4 (3), 253-8, 2012 [Non-patent document 2] TC Laurent and JR Fraser, “Hyaluronan,” FASEB J, Vol. 6, pp.2397-2402, 1992 [Non-patent document 3] Mamoru Kimura, Absorption of Orally Intaken Hyaluronic Acid, Functional Food Research 14:30-35, 2018 Summary of the Invention [Problem to be solved by the invention]

[0008] The molecular weight of typical hyaluronic acid is said to be 800,000 to 1,200,000. However, the molecular weight of the hyaluronic acid used in Non-Patent Document 3 is 300,000, which is smaller than the molecular weight of typical hyaluronic acid. Therefore, it is unclear whether similar degradation and resynthesis will occur when the molecular weight increases. Furthermore, the results in Non-Patent Document 3 were obtained in rats, and it is unclear whether similar behavior will be observed in humans. Furthermore, it is unclear how much of the degraded hyaluronic acid is used for resynthesis. Thus, even if hyaluronic acid is directly ingested orally, it remains unclear whether the lost hyaluronic acid can be replenished.

[0009] In addition, indomethacin and aspirin are used as anti-inflammatory drugs, but their topical use has been associated with side effects such as rash and itching. Furthermore, rosmarinic acid was not sufficiently effective.

[0010] The present invention has been made in consideration of the above problems. That is, the object of the present invention is to provide a hyaluronidase inhibitor with high pharmacological activity that inhibits the activity of hyaluronidase, which decomposes hyaluronic acid, in addition to the method of directly orally taking hyaluronic acid to compensate for the loss of hyaluronic acid. [Means for solving the problem]

[0011] The inventors investigated whether there are any substances that inhibit the activity of hyaluronidase, and discovered that hyaluronidase activity is inhibited by the ingestion of α-ketoglutaric acid, which led to the completion of the present invention.

[0012] To solve the above problems, the present invention is characterized by a hyaluronidase activity inhibitor containing α-ketoglutaric acid as an active ingredient, wherein the concentration of α-ketoglutaric acid is preferably 0.2 to 4 mM.

[0013] According to this configuration, the activity of hyaluronidase can be inhibited, thereby maintaining the amount of hyaluronic acid. [Effects of the Invention]

[0014] According to this invention, the activity of hyaluronidase can be inhibited, thereby preventing the decrease of hyaluronic acid. This allows the amount of hyaluronic acid to be maintained. This is expected to not only prevent skin aging caused by the decrease of hyaluronic acid, but also improve symptoms associated with the decrease of hyaluronic acid in other organs related to hyaluronic acid. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the hyaluronidase activity inhibition rate at each concentration of α-ketoglutaric acid. [Figure 2] FIG. 1 shows the inhibitory effect of α-ketoglutaric acid on hyaluronidase activity in human cells. DETAILED DESCRIPTION OF THE INVENTION

[0016] The hyaluronidase activity inhibitor referred to in the present invention may be any substance that has the effect of inhibiting hyaluronidase activity, and is characterized by containing α-ketoglutaric acid as an active ingredient.

[0017] Here, the "α-ketoglutaric acid" used in the present invention is a type of naturally occurring organic compound known as an intermediate in the citric acid cycle. α-Ketoglutaric acid is also known to be involved in essential physiological processes such as energy production, which is the primary function of food, biosynthesis of specific amino acids, regulation of redox homeostasis, and detoxification of harmful substances. The α-ketoglutaric acid used in the present invention may be extracted and purified from natural products, or may be industrially chemically synthesized.

[0018] However, there are no literature documents describing the absorption rate of α-ketoglutaric acid when ingested. On the other hand, in human studies, oral intake of 3.6 g / day resulted in an increase in blood glutamic acid (a metabolite of ketoglutaric acid) concentrations, and oral administration of 6.0 g / day of calcium α-ketoglutarate was confirmed to have a significant effect on bone mineral density in postmenopausal women. Therefore, in the present invention, the daily intake is preferably 0.001 to 6.0 g, more preferably 0.01 to 6.0 g, and even more preferably 3.6 to 6.0 g.

[0019] α-Ketoglutaric acid may be used as a solid or as a liquid by dissolving it in a solvent, preferably water.

[0020] The form of the hyaluronidase activity inhibitor of the present invention can be either solid or liquid, and can be selected arbitrarily depending on the form in which the hyaluronidase activity inhibitor of the present invention is used.

[0021] Furthermore, the hyaluronidase activity inhibitors of the present invention can be incorporated into foods and beverages. For example, they can be used in dairy products such as fermented milk and lactic acid bacteria drinks, butter, egg products such as mayonnaise, and sweet breads such as butter cake. They can also be suitably used in processed foods such as instant noodles and cookies. In addition to the above, they may be formulated into formulations (e.g., powders, granules, capsules, tablets, etc.) by adding appropriate carriers and additives as needed.

[0022] The hyaluronidase activity inhibitor of the present invention is also useful when incorporated into foods for specified health uses, nutritional supplements, functional foods, etc., in addition to general beverages and foods. [Example]

[0023] The present invention will be described in further detail below, but is not limited thereto. In this example, the inhibition of hyaluronidase activity was evaluated using cultured cells.

[0024] First, the reagents were prepared. Hyaluronidase: Hyaluronidase (Hyaluronidase from bovine tests Type IV-S: manufactured by Sigma-Aldrich) was dissolved in 0.1 M acetate buffer (pH 4.0) and adjusted to 10 mg / mL. Substrate solution: Sodium hyaluronate (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 0.1 M acetate buffer (pH 4.0) and adjusted to 1 mg / mL. Hyaluronidase activator: 20 mg of Compound 48 / 80 and 73.5 mg of calcium chloride dihydrate were dissolved in 10 mL of 0.1 M acetate buffer (pH 4.0) to prepare the solution. p-DAD: 5 g of p-DAD reagent (p-Dimethylaminobenzaldehyde), 6 mL of 10 N HCl, and 44 mL of acetic acid were mixed and diluted 10-fold with acetic acid immediately before use. α-Ketoglutaric acid: α-Ketoglutaric acid (trade name "2-oxoglutaric acid": manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in ion-exchanged water to prepare solutions with concentrations of 1, 11, 12, 13, 14, 15, and 20 mM.

[0025] Hyaluronidase activity inhibition was evaluated as follows. Ten μL of α-ketoglutaric acid adjusted to each concentration and 5 μL of hyaluronidase adjusted to 10 mg / mL were mixed and incubated at 37°C for 20 minutes. Next, 10 μL of the adjusted hyaluronidase activator was added and incubated at 37°C for 20 minutes. 25 μL of the adjusted substrate solution was then added and incubated at 37°C for 40 minutes. To stop the hyaluronidase enzymatic reaction, 10 μL of 0.4 N NaOH and 10 μL of a buffer solution (pH 9.1) adjusted to a final concentration of 0.8 M boric acid and 1 M KOH were added, followed by heating at 95°C for 3 minutes. After incubation at 37°C for 10 minutes, 600 μL of the adjusted p-DAD was added and incubated at 37°C for 20 minutes. After the reaction, 200 μL of the sample solution was taken and the absorbance at 600 nm was measured using a microplate reader. In addition, only the solvent (ion-exchanged water) was added to the control sample.

[0026] The hyaluronidase inhibition rate (%) was calculated using the following formula. Inhibition rate (%)=[1-(S / C)]×100...(1) Here, S is the absorbance of the solution to which each component was added as a sample and corrected with the blank, and C is the absorbance of the solution to which only the solvent was added as a sample and corrected with the blank. Furthermore, the blank is the absorbance of the reaction solution to which each sample was added but no enzyme was added.

[0027] The hyaluronidase inhibition rate (%) of the aqueous α-ketoglutaric acid solution added at each concentration was calculated according to the above formula (1), with the hyaluronidase inhibition rate of ion-exchanged water set at 0%. The results are shown in Figure 1.

[0028] As is clear from Figure 1, when the substrate solution is mixed and the reaction is initiated, the hyaluronidase inhibition rate begins to increase when the α-ketoglutaric acid concentration exceeds 0.2 mM. In particular, at α-ketoglutaric acid concentrations of 2.2 mM to 2.8 mM, the curve showing the inhibition rate rises sharply. It can also be seen that the hyaluronidase inhibition rate exceeds 50% when α-ketoglutaric acid is added at 2.6 mM or higher. On the other hand, at 3 mM or higher, the slope of the curve showing the inhibition rate levels off, suggesting that equilibrium has been reached. These findings suggest that hyaluronidase activity can be inhibited by increasing the α-ketoglutaric acid concentration to 0.2 mM or higher.

[0029] Next, we examined the inhibitory effect of α-ketoglutarate on hyaluronidase activity using normal human dermal fibroblasts (NHDFs). Normal human dermal fibroblasts (NHDFs) were subcultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0030] The cell culture conditions for quantifying hyaluronic acid were as follows: NHDF were cultured in a 35 mm dish at 1.0 × 10 5The cells were seeded at 100 cells / dish and cultured in 10% FBS-DMEM at 37°C and 5% CO2 for 5 days. After 5 days of culture, the culture medium was removed and the cells were washed with 1 mL of phosphate-buffered saline (PBS(-)). 500 μL of PBS(-) was then added. The PBS(-)-added dishes were either not irradiated with UV-B or irradiated with UV-B (lamp: AS ONE Handy UV Lamp LUV-4) at 30 mJ / cm. 2 The cells were then irradiated at an intensity of 100 μM. The PBS(-) was then removed, and 1 mL of DMEM (0% FBS) containing water, sodium cromoglycate (final concentration 100 μM), or α-ketoglutaric acid (final concentration 100 μM) was added to the UV-B-irradiated cells. DMEM with water was designated UV(+), DMEM with sodium cromoglycate was designated UV(+) Cont., and DMEM with α-ketoglutaric acid was designated UV(+) AKG. 1 mL of DMEM (0% FBS) with water was added to the UV-B-unirradiated cells (hereafter referred to simply as "UV(-)"). All cells were cultured for 3 days at 37°C under 5% CO2 conditions. After 3 days of culture, the cell supernatant was collected and diluted 100-fold to prepare the samples. The hyaluronan content of each sample was quantified according to the protocol of the HA ELISA assay kit (R&D Systems Inc., Minneapolis, MN, USA).

[0031] The results are shown in Figure 2. Figure 2 shows the mean ± SD for n = 5 data points. Significant differences between the UV(-) and UV(+) samples, between the UV(+) and UV(+) AKG samples, and between the UV(+) and UV(+) Cont. samples were calculated by t-test with p<0.05 (indicated by "*" in the figure) and p<0.01 (indicated by "**" in the figure). As is clear from Figure 2, when comparing the UV(+) and UV(-) samples in water-added medium, the amount of hyaluronic acid in the UV(+) sample was found to be lower. This suggests that UV irradiation activates hyaluronidase, resulting in the degradation of hyaluronic acid.

[0032] Next, a comparison was made between the UV irradiation groups. It was found that the decrease in hyaluronic acid content was significantly suppressed in sample UV(+)Cont., which contained sodium cromoglycate, known to have strong hyaluronidase inhibitory activity, and sample UV(+)AKG, which contained α-ketoglutaric acid, compared to sample UV(+) which contained water. This suggests that α-ketoglutaric acid inhibits the hyaluronidase activity activated by UV irradiation. Furthermore, it was suggested that the inhibition of hyaluronidase activity suppresses the degradation of hyaluronic acid.

[0033] According to the present invention, the hydrolysis of hyaluronic acid can be prevented and maintained, which is expected to not only prevent skin aging caused by a decrease in hyaluronic acid, but also improve symptoms associated with a decrease in hyaluronic acid in other organs related to hyaluronic acid.

Claims

1. A hyaluronidase activity inhibitor whose active ingredient is α-ketoglutaric acid.

2. The hyaluronidase activity inhibitor according to claim 1, wherein the concentration of α-ketoglutaric acid is 0.2 to 4 mM.

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