Hyaluronic acid production promoter

Abstract

The present invention provides: a novel hyaluronic acid production promoter; and a skin external preparation or a therapeutic agent that promotes the production of a hyaluronic acid. Specifically, by using a magnesium compound as an active ingredient, a hyaluronic acid production promoter, a skin external preparation or a therapeutic agent that promotes the production of a hyaluronic acid are prepared. The present invention can be used for preventing human skin aging (retaining skin tension and elasticity), preventing and treating arthritis, etc., and cosmetological or non-therapeutic methods for the early treatment of burns, etc.

Description

Technical Field

[0001] The present invention relates to a hyaluronic acid production promoter and a cosmetic, beauty or non-therapeutic method using the same.

Background Art

[0002] In recent years, research on aging has been progressing. Macroscopically, aging is an important factor in skin aging. In addition, factors such as dryness, oxidation, sunlight (ultraviolet rays), etc. are also cited as direct factors related to skin aging. Specific phenomena of skin aging include the decrease of mucopolysaccharides such as hyaluronic acid, the cross-linking reaction of collagen, and cell damage by ultraviolet rays.

[0003] Among them, hyaluronic acid has many functions such as retaining moisture in the intercellular space, retaining cells based on forming a jelly-like matrix in the tissue, retaining the lubricity and flexibility of the tissue, resisting external forces such as mechanical damage, and preventing bacterial infection. For example, it is said that the amount of hyaluronic acid in the skin decreases with aging, and accordingly, skin aging such as fine wrinkles and roughness appears. Therefore, many cosmetics containing hyaluronic acid or collagen have been proposed as improving agents for such aging skin. However, these conventional cosmetics only exhibit a moisturizing effect on the skin surface and do not essentially improve aging skin. In addition, cosmetics containing various vitamins and herbal medicines as skin cell activators have been proposed, but at present, they have not yet reached the level of being able to improve and treat aging skin.

[0004] Furthermore, hyaluronic acid contained in synovial fluid coats the surface of articular cartilage and contributes to the smooth operation of joint function. The hyaluronic acid concentration in synovial fluid of a normal person is approximately 2.3 mg / mL, but in the case of rheumatoid arthritis, the hyaluronic acid concentration in synovial fluid decreases to approximately 1.2 mg / mL, and at the same time, the viscosity of the synovial fluid also decreases significantly (Non-Patent Literature 1). It is also known that a decrease in hyaluronic acid content occurs in septic arthritis and gouty arthritis, similar to the case of rheumatoid arthritis (Non-Patent Literature 2). In the above diseases, increasing the amount of hyaluronic acid in synovial fluid is considered in order to improve lubrication function, cover and protect articular cartilage, suppress pain, and improve or normalize pathological synovial fluid. For example, it has been reported that the above improvements are observed when sodium hyaluronate is injected into the joint in patients with rheumatoid arthritis (Non-Patent Literature 3). Similarly, the above improvement effects have been reported with hyaluronic acid injection therapy in the joint in traumatic arthritis, osteoarthritis, and osteoarthritis (Non-Patent Literature 4).

[0005] However, treatment for the above-mentioned diseases is long-term and requires a doctor's prescription. Therefore, there has been a need for a topical skin preparation containing a hyaluronic acid production promoter that can be easily used in daily life.

[0006] Furthermore, it is known that during the healing process after a burn injury, hyaluronic acid significantly increases in granulation tissue from the initial stage when granulation tissue proliferates from below the necrotic tissue until the entire tissue is replaced by granulation tissue (Non-Patent Literature 5). Therefore, hyaluronic acid production promoters are also expected to be useful as early treatments for burns.

[0007] On the other hand, known agents that promote hyaluronic acid production in human cells include cytokines such as insulin-like growth factor-1, epidermal growth factor (Non-Patent Literature 6), and interleukin-1 (Non-Patent Literature 7), or phorbol esters (Non-Patent Literature 8). However, none of these can be easily and safely used as cosmetics, bath additives, or pharmaceuticals.

[0008] On the other hand, it has been reported that a skin barrier function recovery promoter consisting of magnesium salts and calcium salts can restore skin barrier function in a very short time, thereby preventing epidermal proliferation abnormalities and other problems caused by a decline in epidermal function (Patent Document 1). However, it was previously unknown that magnesium compounds have the effect of increasing hyaluronic acid levels in human cells. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2000-290135 [Non-patent literature]

[0010] [Non-Patent Document 1] “Arthritis Rheumatism”, vol.10, p.357(1967) [Non-Patent Document 2] "Bond Composition," Kinbara Publishing, 481 pages, 1984. [Non-Patent Document 3] "Inflammation," Japanese Society for Inflammation, Vol. 11, p. 16, 1991. [Non-Patent Document 4] "Connective Tissue and Disease," Kodansha, p. 246, 1980. [Non-Patent Document 5] "Connective Tissue and Disease," Kodansha, p. 153, 1980. [Non-Patent Document 6] “Biochemica Biophysica Acta”, 1014, p.305(1989) [Non-Patent Document 7] Journal of the Japan Society of Obstetrics and Gynecology, Vol. 41, p. 1943, 1989. [Non-Patent Document 8] “Experimental Cell Research”, vol.148, p.377(1983) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The present invention has been made in view of the above circumstances, and aims to provide a hyaluronic acid production promoter that increases the amount of hyaluronic acid by promoting hyaluronic acid production ability. [Means for solving the problem]

[0012] The inventors investigated the effects of magnesium on the gene expression of factors involved in skin differentiation and moisturizing using human keratinocyte-derived HaCaT cells, and for the first time discovered that magnesium compounds have excellent hyaluronic acid production-promoting effects. They then invented a hyaluronic acid production-promoting agent characterized by containing a magnesium compound as an active ingredient.

[0013] Therefore, the present invention relates to the following invention: [1] A hyaluronic acid production promoter characterized by containing a magnesium compound as an active ingredient. [2] The hyaluronic acid production promoter described in item 1, wherein the magnesium compound is magnesium chloride, magnesium lactate, or magnesium sulfate. [3] Hyaluronic acid production promoters as described in item 1 or 2, used to promote the production of hyaluronic acid in epidermal cells and / or keratinocytes. [4] A cosmetic or non-therapeutic method comprising applying a hyaluronic acid production promoter containing a magnesium compound as an active ingredient. [5] Use of magnesium compounds to promote hyaluronic acid production. [6] A topical skin preparation that promotes the production of hyaluronic acid, characterized by containing a magnesium compound as an active ingredient. [7] A therapeutic agent for treating diseases caused by abnormal degradation of hyaluronic acid or decreased hyaluronic acid production, characterized by containing a magnesium compound as an active ingredient. [8] A method for screening compounds that promote the production of hyaluronic acid, A step of bringing cells into contact with the compound, A step of measuring the intracellular magnesium concentration, A step of determining the presence or absence of an increase in the intracellular magnesium concentration depending on the presence or absence of the compound A method comprising the above. [9] The method according to item 8, further comprising a step of measuring a change in the production amount of hyaluronic acid. [Effect of the Invention]

[0014] The production of hyaluronic acid in cells can be promoted using a magnesium compound. Furthermore, by promoting the production of hyaluronic acid, it can be effectively applied to preventing the aging of human skin (maintaining skin firmness and elasticity), preventing and treating arthritis, etc., and treating burns in the early stage. [Brief Description of the Drawings]

[0015] [Figure 1] Figure 1 shows the mechanism by which the uptake of a magnesium compound promotes the expression of hyaluronic acid synthase (HAS) and the production of hyaluronic acid in cells. [Figure 2] Figure 2 shows the mechanism by which the uptake of a magnesium compound promotes the expression of hyaluronic acid synthase (HAS) and the production of hyaluronic acid in cells. [Figure 3] Figure 3 shows the promotion of the expression of hyaluronic acid synthase (HAS) by a magnesium compound. Exposure of HaCaT cells to a high-magnesium medium promotes the expression of HAS2 and HAS3, and exposure to a low-magnesium medium decreases the expression of HAS2 and HAS3. [Figure 4] Figure 4 shows the results of quantitatively promoting the expression level of hyaluronic acid synthase (HAS) by a magnesium compound. Exposure of HaCaT cells to a high-magnesium medium increases the relative protein amounts of HAS2 and HAS3, and exposure to a low-magnesium medium decreases the relative protein amounts of HAS2 and HAS3. In the figure, C on the horizontal axis represents control, H represents high magnesium concentration, and L represents low magnesium concentration. [Figure 5]A shows the result that magnesium compounds promote hyaluronic acid synthesis. It shows that exposure of HaCaT cells to a high-magnesium medium increases extracellular hyaluronic acid levels, while exposure to a low-magnesium medium decreases them. On the horizontal axis, C represents the control, H represents high magnesium concentration, and L represents low magnesium concentration. B shows the results of investigating the effect of NIPAL2 and NIPAL4 on the uptake of magnesium compounds on the cell surface. It shows that introduction of NIPAL2siRNA and NIPAL4siRNA suppresses the uptake of magnesium compounds and inhibits the production of extracellular hyaluronic acid. On the horizontal axis, Neg represents the negative control, N2 represents the negative control when exposed to 5 mM MgCl2, N4 represents the NIPAL2siRNA-introduced sample, and N4 represents the NIPAL4 siRNA-introduced sample. [Figure 6] Figure 6 shows the results of measuring the increase in HAS2 and HAS3 mRNA levels when each magnesium compound was added. An increase in HAS2 and HAS3 mRNA levels was observed with all three compounds: magnesium chloride (MgCl2), magnesium lactate (Mg-lac), and magnesium sulfate (MgSO4). [Modes for carrying out the invention]

[0016] In one embodiment of the present invention, the present invention relates to a hyaluronic acid production promoter characterized by containing a magnesium compound as an active ingredient.

[0017] Genetic mutations in NIPAL4, which functions as a magnesium channel, have been identified in patients with hereditary ichthyosis, and a link between skin damage and abnormal magnesium transport has been suggested. However, the role and mechanism of action of magnesium compounds in maintaining the skin barrier remained unclear. The inventors of this invention investigated the effect of magnesium on the gene expression of factors involved in skin differentiation and moisturizing using human keratinocyte-derived HaCaT cells, and found that the expression level of hyaluronic acid synthase (HAS) changed, so they investigated the mechanism of action.

[0018] The inventors have for the first time discovered that the expression level of hyaluronic acid synthase (HAS) and the production of hyaluronic acid are promoted by using magnesium compounds. When the relationship between HAS and NIPAL2 and NIPAL4 was investigated, the introduction of NIPAL2 / 4siRNA reduced the amount of HAS2 / 3 mRNA, and the increase in HAS2 / 3 mRNA levels due to exposure to a high-magnesium medium was suppressed. Therefore, it was suggested that NIPAL2 and NIPAL4 function as magnesium influx channels. It is thought that magnesium ions are taken up via NIPAL2 / 4 on the cell surface, thereby promoting the expression of HAS2 / 3 and the production of hyaluronic acid in cells (Figure 1).

[0019] To elucidate the regulatory mechanism of HAS2 / 3 expression by magnesium, we investigated its effect on the phosphorylation of cell signaling factors. Further investigation of reporter activity revealed that the mechanism shown in Figure 2 is involved in Mg-mediated HA production. Therefore, by using magnesium compounds, the production of hyaluronic acid can be promoted.

[0020] In this specification, a hyaluronic acid production promoter means an agent that has the effect of promoting the production of hyaluronic acid by cells and / or the ability to produce hyaluronic acid. The effect of promoting hyaluronic acid production and / or hyaluronic acid production ability is evaluated by measuring the amount of extracellular hyaluronic acid produced after culturing cells in a culture medium containing a hyaluronic acid production promoter.

[0021] Examples of magnesium compounds used in the hyaluronic acid production promoter of the present invention include magnesium-containing compounds such as halides and double halides, oxides and related oxide compounds, sulfides, nitrides, carbides, oxyacid salts, and organomagnesium compounds. Particularly preferred are magnesium chloride, magnesium lactate, or magnesium sulfate.

[0022] The hyaluronic acid production promoter of the present invention is used, for example, to promote the production of hyaluronic acid in epidermal cells, dermal fibroblasts, and / or keratinocytes, but is not limited to these. In the present invention, the effect is particularly pronounced in epidermal cells and / or keratinocytes.

[0023] One aspect of the present invention relates to a topical skin preparation that promotes the production of hyaluronic acid, characterized by containing a magnesium compound as an active ingredient. The hyaluronic acid production promoter of the present invention, by using a magnesium compound, promotes the production of hyaluronic acid and can be effectively applied to prevent aging of human skin (maintaining skin firmness and elasticity), prevent and treat arthritis, and treat burns in their initial stages. Therefore, a topical skin preparation (including pharmaceuticals, quasi-drugs, and cosmetics) that promotes the production of hyaluronic acid and / or a therapeutic agent for treating diseases caused by abnormal degradation of hyaluronic acid or a decrease in hyaluronic acid production capacity are provided. Furthermore, one aspect of the present invention relates to a therapeutic agent for treating diseases caused by abnormal degradation of hyaluronic acid or decreased hyaluronic acid production, characterized by containing a magnesium compound as an active ingredient. Diseases include, but are not limited to, hepatitis, gingivitis, rheumatism, osteoarthritis, malignant tumors, cirrhosis, transplant refusal, scleroderma, arteriosclerosis, fibrosis, xerosis, dry skin, rough skin, nephritis, keloids, over-repair, and sepsis. Moreover, one aspect of the present invention relates to a method for treating diseases caused by abnormal degradation of hyaluronic acid or decreased hyaluronic acid production in a subject, comprising administering a composition containing a magnesium compound as an active ingredient to the subject. The subject may be a mammal including humans or a non-human mammal, but is preferably human.

[0024] When the magnesium compound of the present invention is incorporated into a topical skin preparation or therapeutic agent, the magnesium compound may be included in an amount of, for example, 1 to 20 mM or 0.000001 to 1.0% by mass of the total amount of the topical preparation.

[0025] When applying the present invention to a topical skin preparation or therapeutic agent (composition), in addition to the above components, other components commonly used in topical skin preparations such as cosmetics and pharmaceuticals may be added as needed, within the limits that do not impair the effects of the present invention, for example, antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc.

[0026] Furthermore, metal ion sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; preservatives such as methylparaben, ethylparaben, and butylparaben; caffeine, tannins, verapamil, tranexamic acid and its derivatives; licorice extract, glabridin, hot water extract of quince fruit, various herbal medicines; drugs such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts; whitening agents such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, and kojic acid; and sugars such as glucose, fructose, mannose, sucrose, and trehalose can also be added as appropriate.

[0027] Topical skin preparations can be applied to the skin, such as cosmetics and quasi-drugs, and are particularly suitable for a wide range of cosmetic applications. Any dosage form that can be applied to the skin is acceptable, including solutions, solubilization, emulsification, powder dispersion, water-oil two-layer systems, water-oil-powder three-layer systems, ointments, lotions, gels, aerosols, and any other dosage form.

[0028] The form of use is also arbitrary; for example, it can be used in facial cosmetics such as lotions, emulsions, creams, and masks, as well as makeup cosmetics such as foundations, lipsticks, and eyeshadows, fragrance cosmetics, bath additives, etc.

[0029] Furthermore, it can be widely applied to makeup cosmetics such as foundations, and to toiletry products such as body soaps and bar soaps. In addition, it can be widely applied to quasi-drugs such as various ointments. Moreover, the forms that the hyaluronic acid production promoter of the present invention can take are not limited to these dosage forms and shapes.

[0030] When incorporating the hyaluronic acid production promoter of the present invention into food, beverages, or animal feed, the amount of magnesium compound can be appropriately determined according to the type, purpose, form, and method of use. For example, it can be included in the total amount of tablets or capsules at 1 to 90% by mass, and in other foods and beverages at 0.001 to 50% by mass. It is preferable to prepare the product so that the daily intake of magnesium compound for adults is approximately 1 to 370 mg. In particular, when used as a health food or beverage, it is preferable to include the active ingredient of the present invention in an amount that allows the desired effect to be fully exerted.

[0031] Food and beverage products and animal feeds can be molded into any form, such as granules, pellets, paste, gel, solid, or liquid. These can contain various known substances permitted for inclusion in food and beverage products, such as binders, disintegrants, thickeners, dispersants, reabsorption enhancers, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers, and pH adjusters.

[0032] When the hyaluronic acid production promoter of the present invention is used as a pharmaceutical preparation, the preparation may be administered orally or parenterally (intravenous administration, intraperitoneal administration, etc.) as appropriate. The dosage form is also arbitrary, and any of the following forms can be appropriately prepared by known methods: for example, oral solid preparations such as tablets, granules, powders, and capsules; oral liquid preparations such as oral solutions and syrups; or parenteral liquid preparations such as injections. These pharmaceutical preparations may also contain excipients such as binders, disintegrants, thickeners, dispersants, reabsorption enhancers, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers, and pH adjusters as appropriate.

[0033] When the hyaluronic acid production promoter of the present invention is used as a topical skin preparation, food or beverage, animal feed, pharmaceutical preparation, etc., it is useful for the treatment, prevention, and improvement of various symptoms, diseases, and pathological conditions involving non-enzymatic protein glycation. Specific examples of applications include, for instance, preventing aging of human skin (maintaining skin firmness and elasticity), preventing and treating arthritis, and treating burns in their initial stages. Furthermore, it can be applied to topical skin preparations, functional foods and beverages, foods for the sick, and foods for specified health uses, etc., based on the concept of its physiological functions such as the treatment, prevention, and improvement of the above-mentioned symptoms, diseases, and pathological conditions, and can be indicated accordingly.

[0034] One aspect of the present invention relates to a cosmetic or non-therapeutic method comprising applying a hyaluronic acid production promoter containing a magnesium compound as an active ingredient.

[0035] The hyaluronic acid production promoters, topical skin preparations, or therapeutic agents disclosed herein can be used in cosmetic or non-therapeutic methods for preventing aging of human skin (maintaining skin firmness and elasticity), preventing and treating arthritis, and treating burns in their initial stages. In the cosmetic or non-therapeutic methods of the present invention, the topical skin preparation composition is applied to the area where the desired effect is to be obtained, such as the face, neck, hands, arms, legs, etc. Application can be carried out in any manner, such as by hand or with an applicator. The number of applications, frequency, amount, etc., are set as appropriate according to the desired effect. Note that the cosmetic or non-therapeutic methods of the present invention do not include so-called medical procedures.

[0036] Furthermore, one aspect of the present invention relates to the use of magnesium compounds to promote hyaluronic acid production. Data disclosed herein demonstrate that magnesium compounds have excellent hyaluronic acid production capabilities. Therefore, magnesium compounds can be used to prepare hyaluronic acid production promoters, topical skin preparations that promote hyaluronic acid production, and therapeutic agents that treat diseases caused by reduced hyaluronic acid production. That is, one aspect of the present invention also relates to the use of magnesium compounds in the manufacture of these topical skin preparations and therapeutic agents.

[0037] Furthermore, one aspect of the present invention is a method for screening compounds that promote the production of hyaluronic acid, A step of bringing cells into contact with the compound, A process for measuring intracellular magnesium concentration, A step to determine whether or not there is an increase in intracellular magnesium concentration due to the presence or absence of the compound. Regarding methods including Hyaluronic acid production capacity can be measured by measuring the intracellular magnesium compound concentration. Specifically, since hyaluronic acid concentration increases with increasing magnesium ion concentration, candidate compounds can be selected in the screening of compounds that promote hyaluronic acid production by determining whether or not the intracellular magnesium concentration increases depending on the presence or absence of the candidate compound. Magnesium concentration is evaluated by measuring the intracellular magnesium concentration after culturing cells in a culture medium containing the candidate compound.

[0038] Compounds to be screened may include those found in compound libraries or libraries of extracts from microorganisms, plants, animals, etc. Intracellular magnesium concentration can be measured, for example, by fluorescence measurement using a magnesium fluorescent probe. Examples of magnesium fluorescent probes include, but are not limited to, KMG-20-AM, available from Fujifilm Wako Pure Chemical Corporation, Mag-Fura-2, and Magnesium Green (trademark), available from Thermo Fisher Scientific.

[0039] Furthermore, one aspect of the present invention relates to the screening method described above, further comprising the step of measuring a change in the amount of hyaluronic acid produced. In the above screening method, candidate compounds can be selected by measuring the change in the amount of hyaluronic acid produced.

[0040] Changes in hyaluronic acid production can be determined, for example, by measuring the amount of extracellular hyaluronic acid (HA) using known methods such as ELISA. Alternatively, changes in hyaluronic acid production may be measured based on changes in the expression level of hyaluronic acid synthase (HAS) (HAS2 / 3 mRNA level or HAS2 / 3 protein level).

[0041] All references made herein are incorporated herein by citation in their entirety.

[0042] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0043] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0044] Example 1: Identification of HAS2 and HAS3 expression levels using magnesium compounds When intracellular magnesium concentration ([Mg2+]) was measured using fluorescence chromatography, exposure of HaCaT cells to a high-magnesium medium resulted in increased intracellular magnesium concentration and an increase in HAS2 and HAS3 mRNA levels. At low magnesium concentrations, intracellular magnesium concentration decreased, and a decrease in HAS2 and HAS3 mRNA levels was observed.

[0045] The results are shown in Figures 3 and 4. As can be seen from the results in Figure 3, high magnesium concentrations promoted the expression of HAS2 and HAS3. Figure 4 similarly shows that high magnesium concentrations also increased the relative protein levels of HAS2 and HAS3.

[0046] Example 2: Measurement of hyaluronic acid content by magnesium concentration Hyaluronic acid produced extracellularly (in the culture medium) was measured by ELISA (competitive assay) using a hyaluronic acid measurement kit (PG Research). Figure 5A shows that exposure of HaCaT cells to a high-magnesium medium increases the amount of extracellular hyaluronic acid, while exposure to a low-magnesium medium decreases the amount of extracellular hyaluronic acid. When the relationship between HAS expression and NIPAL2 and NIPAL4 was investigated, the introduction of NIPAL2 siRNA and NIPAL4 siRNA reduced HAS2 / 3 mRNA levels and suppressed the increase in HAS2 / 3 mRNA levels caused by exposure to a high-magnesium medium. Figure 5B shows that the introduction of NIPAL2 siRNA and NIPAL4 siRNA suppressed the uptake of magnesium compounds and inhibited the production of extracellular hyaluronic acid. This suggests that NIPAL2 and NIPAL4 function as magnesium influx channels.

[0047] Example 3: Effect of magnesium compounds on HAS2 / 3 mRNA levels HaCaT cells were cultured for 4 days, then incubated for 6 hours in a medium supplemented with 5 mM magnesium chloride (MgCl2), magnesium lactate (Mg-lac), and magnesium sulfate (MgSO4). After RNA extraction, HAS2 / 3 mRNA levels were measured by real-time PCR. The results are shown in Figure 6. An increase in HAS2 and HAS3 mRNA levels was observed with all three: magnesium chloride (MgCl2), magnesium lactate (Mg-lac), and magnesium sulfate (MgSO4).

[0048] As is clear from the results in Figures 3-6, magnesium ion uptake was found to promote HAS2 / 3 expression and hyaluronic acid production.

Claims

1. A hyaluronic acid production promoter characterized by containing a magnesium compound as an active ingredient, A hyaluronic acid production promoter wherein the magnesium compound is magnesium chloride, magnesium lactate, or magnesium sulfate.

2. A hyaluronic acid production promoter according to claim 1, used to promote the production of hyaluronic acid in epidermal cells and / or keratinocytes.

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