Defensin expression promoter

Abstract

To provide a defensin expression promoter having an excellent defensin expression promoting action on the skin or in vivo.SOLUTION: Lysophosphatidic acid or its salt is used as an active ingredient. Since lysophosphatidic acid or a salt thereof has an excellent defensin expression promoting action on the skin or in vivo, it can be preferably used for the purpose of improving the antibacterial action on the skin or in vivo. It is particularly effective for promoting the expression of human β - defensin-1 and human β - defensin-3 among defensins. In addition, since lysophosphatidic acid or a salt thereof also has an action of suppressing proliferation of Propionibacterium acnes based on the action of promoting expression of human β - defensin-3, it can be used as an agent for suppressing proliferation of Propionibacterium acnes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a defensin expression promoter containing lysophosphatidic acid or a salt thereof, and a composition containing the promoter for improving antibacterial activity on the skin or in the body.The present invention also relates to an acne bacteria growth inhibitor containing lysophosphatidic acid or a salt thereof, and a composition containing the inhibitor for preventing or ameliorating acne.

[0002] It is known that various antibacterial substances (antimicrobial peptides) are produced in vivo as part of the innate immune mechanism of living organisms. Antibacterial peptides exert strong antibacterial activity against various microorganisms, including bacteria, fungi, and viruses, and play roles in local infection defense and maintaining the homeostasis of resident bacteria. Therefore, promoting the expression of antibacterial peptides in vivo could lead to improved immunity against microbial invasion and the treatment of infectious diseases and other diseases.

[0003] Among antimicrobial peptides, defensins are one of the most widely studied. In humans, α-defensins, which are primarily found in phagocytes such as neutrophils, and β-defensins, which are primarily found in epithelial cells, including skin, are known to be representative. Currently, four major types of human β-defensins (hBDs), hBD-1 to hBD-4, are known. While hBD-1 is constitutively expressed in the skin, hBD-2 and hBD-3 have been reported to be inducible antimicrobial peptides whose expression is induced by bacterial infection or inflammatory stimuli, and are thought to be closely related to various infections and inflammation. In the skin, their relationship with the onset and aggravation of atopic dermatitis has been suggested (Non-Patent Document 1). Furthermore, among human β-defensins, hBD-3 is known to have a strong antibacterial effect against Propionibacterium acnes and to be effective in preventing or improving acne (Patent Document 1, Non-Patent Document 2).

[0004] Phospholipids are a general term for compounds with a structure consisting of a glycerol backbone with fatty acids ester-linked at the sn-1 and sn-2 positions and a polar group containing phosphate at the sn-3 position. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA) are known depending on the polar group structure. Lysophospholipids are compounds formed by hydrolysis of one of the two fatty acids bound to a phospholipid. Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and lysophosphatidic acid (LPA) are known. In recent years, extensive research has been conducted on these various phospholipids and lysophospholipids, focusing on their individual functions. Among these, lysophosphatidic acid has also been widely studied, and it has been reported to have physiologically active effects such as promoting collagen production (Patent Document 2), promoting aquaporin gene expression (Patent Document 3), promoting ceramide production, and promoting tight junction formation (Non-Patent Document 3). However, its effect of promoting defensin expression is unknown. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2018-184431 [Patent Document 2] International Publication No. 2009 / 028220 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-063515 [Non-patent literature]

[0006] [Non-Patent Document 1] FRAGRANCE JOURNAL October 2006 issue, p.99~104 [Non-patent document 2] IFSCC Magazine Vol.18, No.1(2005), p.5~10 [Non-patent document 3] FRAGRANCE JOURNAL May 2009 issue, p.31~34 Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to provide a defensin expression promoter that has an excellent defensin expression promoting effect in the skin or in the living body. [Means for solving the problem]

[0008] As a result of extensive research conducted by the present inventors to solve the above problems, they discovered that lysophosphatidic acid or a salt thereof has an excellent effect of promoting defensin expression, and thus completed the present invention. [Effects of the Invention]

[0009] Lysophosphatidic acid or a salt thereof has an excellent effect of promoting defensin expression in the skin or in the body and can be used as a defensin expression promoter. It is particularly effective in promoting the expression of defensins, particularly human β-defensin-1 and human β-defensin-3. Defensins are antimicrobial peptides that exhibit strong antimicrobial activity against various microorganisms, including bacteria, fungi, and viruses. Therefore, the defensin expression promoter of the present invention can be used to improve antimicrobial activity in the skin or in the body. Furthermore, lysophosphatidic acid or a salt thereof also has the effect of inhibiting the growth of Propionibacterium acnes due to its effect of promoting the expression of human β-defensin-3, and can therefore be used as an Propionibacterium acnes growth inhibitor. Because the growth of Propionibacterium acnes is one of the factors that worsen acne, the Propionibacterium acnes growth inhibitor of the present invention can be used to prevent or improve acne. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a defensin expression promoter containing lysophosphatidic acid or a salt thereof, and a composition containing the promoter for improving antibacterial activity on the skin or in the body. The present invention also relates to an acne bacteria growth inhibitor containing lysophosphatidic acid or a salt thereof, and a composition containing the inhibitor for preventing or ameliorating acne.

[0011] The lysophosphatidic acid used in the present invention is a compound in which one of the two fatty acids bonded to phosphatidic acid, a type of phospholipid, has been hydrolyzed. More specifically, it is a compound represented by the following general formula (1) or (2): [ka] (In the formula, R1 represents a saturated or unsaturated aliphatic acyl group having 14 to 24 carbon atoms.) [ka] (wherein R2 represents a saturated or unsaturated aliphatic acyl group having 14 to 24 carbon atoms).

[0012] Specific examples of the saturated or unsaturated aliphatic acyl group having 14 to 24 carbon atoms for R1 in general formula (1) or R2 in general formula (2) include residues derived from saturated fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and lignoceric acid; and unsaturated fatty acids such as myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid, and are preferably residues derived from palmitic acid, stearic acid, oleic acid, linoleic acid, or linolenic acid.

[0013] The method for producing lysophosphatidic acid of the present invention is not particularly limited, and lysophosphatidic acid produced by a commonly known method can be used. Specifically, the lysophosphatidic acid can be produced by reacting phospholipase A1 or phospholipase A2 with phosphatidic acid in water or a mixture of water and an organic solvent.

[0014] When phosphatidic acid is reacted with phospholipase A2, lysophosphatidic acid of general formula (1) is produced as the main reaction product due to the specificity of the enzyme, but lysophosphatidic acid of general formula (2) is also produced in part. When phospholipase A1 is reacted with phosphatidic acid, lysophosphatidic acid of general formula (2) is produced as the main reaction product due to the specificity of the enzyme, but lysophosphatidic acid of general formula (1) is also produced in part. Furthermore, lysophosphatidic acid of general formula (2) is easily converted to lysophosphatidic acid of general formula (1) over time by rearrangement of the acyl group. In the present invention, lysophosphatidic acid of general formula (1) or lysophosphatidic acid of general formula (2) can be used alone, but as described above, according to general production methods, lysophosphatidic acid of general formula (1) and lysophosphatidic acid of general formula (2) are obtained as a mixture, and therefore, it is preferable to use such lysophosphatidic acid in the present invention.

[0015] The phosphatidic acid used in the production of lysophosphatidic acid used in the present invention may be one synthesized from α-glycerophosphate and a fatty acid, or one produced by reacting phosphatidylcholine with phospholipase D in water or a mixture of water and an organic solvent. The phosphatidylcholine used in the production of phosphatidic acid may be one synthesized from α-glycerophosphatidylcholine and a fatty acid, or a phosphatidylcholine-containing lecithin obtained from soybean, rapeseed, sunflower, safflower, peanut, cottonseed, corn, rice, barley, egg yolk, etc., or a hydrogenated product thereof.

[0016] From the viewpoints of economy and consumer preference, the lysophosphatidic acid used in the present invention is preferably lysophosphatidic acid obtained by treating phosphatidylcholine-containing lecithin or a hydrogenated product thereof with phospholipase D, followed by the treatment of the resulting phosphatidic acid with phospholipase A1 or phospholipase A2. Depending on the type of lecithin used as a raw material, lysophosphatidic acid obtained by such a method may contain, in addition to lysophosphatidic acid, lysophospholipids such as lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, and lysophosphatidylinositol, and may further contain non-lysophospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, depending on the degree of purification. As the lysophosphatidic acid used in the present invention, those containing various components derived from lecithin as described above in addition to lysophosphatidic acid can also be preferably used.

[0017] The lysophosphatidic acid used in the present invention can be in the form of salt.Specifically, it can be metal salt such as sodium, potassium, aluminum, calcium, barium, magnesium, iron, zinc, etc.; organic salt such as ammonium, triethylamine, monoethanolamine, diethanolamine, triethanolamine, pyridine, imidazole, guanidine, lysine, arginine, histidine, ornithine, etc.Among these, sodium, potassium, aluminum, zinc, ammonium, triethylamine, triethanolamine, arginine, etc. are preferred.

[0018] The lysophosphatidic acid or a salt thereof used in the present invention may be used as it is, or may be used as a premix composition in which it is dissolved or dispersed in a common base such as water, gel, polyhydric alcohol, petrolatum, paraffin, ester oil, silicone oil, etc. When using a premix composition, it is more preferable to use a premix composition in which it is dissolved or dispersed in advance in water and / or a polyhydric alcohol, from the viewpoint of ease of handling and improving solubility and dispersibility in water or other media.

[0019] The lysophosphatidic acid or a salt thereof used in the present invention may be a commercially available product containing lysophosphatidic acid or a salt thereof, such as PrimeLipid ALPA (Nippon Fine Chemical Co., Ltd.) or NIKKOL LECINOL MFL (Nikko Chemicals Co., Ltd.).

[0020] The present invention has discovered that lysophosphatidic acid or a salt thereof has the effect of promoting defensin expression. Therefore, lysophosphatidic acid or a salt thereof can be preferably used as an active ingredient in a defensin expression promoter. The amount of lysophosphatidic acid or a salt thereof incorporated into a defensin expression promoter is not particularly limited, and may be incorporated in an amount of 0.005 to 99% by weight, preferably approximately 0.01 to 90% by weight. Because defensins are antimicrobial peptides that exhibit strong antimicrobial activity against various microorganisms, including bacteria, fungi, and viruses, the defensin expression promoter of the present invention can be used to enhance antimicrobial activity on the skin or in vivo. Specifically, the defensin expression promoter of the present invention can be used to improve immunity against microbial invasion, maintain homeostasis of resident flora, and treat diseases such as infectious diseases. In particular, in the skin, it can be preferably used to prevent or treat atopic dermatitis and can also be used to maintain healthy skin.

[0021] The defensin expression promoter of the present invention is effective in promoting the expression of human beta-defensins, which are present mainly in epithelial cells including the skin, and is particularly effective in promoting the expression of human beta-defensin-1 (hBD-1) and human beta-defensin-3 (hBD-3).

[0022] As described in the Background Art section, human β-defensin-3 is known to have a strong antibacterial effect against Propionibacterium acnes. Therefore, lysophosphatidic acid or a salt thereof used in the present invention has the effect of inhibiting the proliferation of Propionibacterium acnes due to its effect of promoting the expression of human β-defensin-3, and can therefore be used as an Propionibacterium acnes growth inhibitor. The amount of lysophosphatidic acid or a salt thereof incorporated into the Propionibacterium acnes growth inhibitor is not particularly limited, and may be in the range of 0.005 to 99% by weight, preferably 0.01 to 90% by weight. Because the proliferation of Propionibacterium acnes is one of the factors that worsen acne, the Propionibacterium acnes growth inhibitor of the present invention can be used for the purpose of preventing or ameliorating acne.

[0023] The defensin expression promoter or the acne bacteria proliferation inhibitor of the present invention can be used in combination with a component having similar physiological activity, which can synergistically exert the effects of the present invention.

[0024] The defensin expression promoter or acne bacteria proliferation inhibitor of the present invention may consist of the active ingredient itself, or may contain, in addition to the active ingredient, various physiologically acceptable bases, carriers, or additives, as well as other medicinal ingredients. The bases, carriers, and additives that can be used are not particularly limited as long as they are those commonly used to obtain the various dosage forms described below, and known agents such as excipients, colorants, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, moisturizers, and pH adjusters may be appropriately selected and used.

[0025] The defensin expression promoter or acne bacteria proliferation inhibitor of the present invention can be administered to a living body by oral administration, injection, transdermal administration, etc. Furthermore, the defensin expression promoter or acne bacteria proliferation inhibitor of the present invention can be used in various dosage forms depending on the intended administration method. Specific examples include oral internal preparations such as tablets, capsules, granules, powders, liquids, and suspensions; parenteral internal preparations such as drip infusions and injections; and external preparations such as dermatological preparations, patches, eye drops, nasal drops, oral preparations, and suppositories. Furthermore, for oral administration, the agent can be incorporated into general foods or health foods, and for external administration, it can be incorporated into cosmetics and the like. The dosage is not particularly limited as long as it is an amount that can achieve the effects of the present invention, and can be appropriately adjusted depending on the dosage form of the preparation, the application site, age, sex, etc. [Example]

[0026] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way.

[0027] Synthesis Example 1: Synthesis of sodium lysophosphatidic acid A 200 mL reaction vessel equipped with a stirrer, thermometer, gas inlet, and condenser was charged with 21 g of soybean lecithin (SLP-PC70, Tsuji Oil Mills), 23.8 g of n-heptane, and 31.5 g of ethyl acetate, and stirred to dissolve at 40°C. Next, 0.7 g of phospholipase D (Meito Sangyo Co., Ltd.), 36.4 g of 1.0 M sodium acetate buffer (pH 6.5), and 0.21 g of calcium chloride were added and stirred at 40°C for 20 hours. After the reaction, 16.6 g of ethanol was added and stirred for 0.5 hours. 22.5 g of 10 wt% aqueous sodium chloride solution was then added and stirred, then allowed to stand and allowed to separate. 280 g of acetone was added to the resulting upper layer, and the precipitated solid was filtered and dried to obtain a sodium phosphatidate-containing product as a yellow viscous solid. A 200 mL reaction vessel equipped with a stirrer, thermometer, gas inlet tube, and condenser was charged with 14.0 g of the yellow viscous solid obtained above, 34.0 g of n-heptane, and 22.5 g of ethyl acetate, and stirred and dissolved at 40 °C. Next, 0.21 g of phospholipase A2 (Nagase 10P / R, Nagase Chemtec), 7.0 g of 1.0 M sodium acetate buffer (pH 6.5), and 0.14 g of calcium chloride were added and stirred at 40 °C for 20 hours. After the reaction, 36.4 g of ethanol was added and stirred for 0.5 hours, followed by 25.9 g of 10 wt% aqueous sodium chloride solution, stirring, and then allowed to stand for separation. 415 g of acetone was added to the resulting lower layer, and the precipitated solid was filtered and dried to obtain a sodium lysophosphatidate-containing product as a yellow solid (yield 45%). The lysophosphatidic acid content was 25.8% by mass. The fatty acid composition was palmitic acid 30%, stearic acid 7%, oleic acid 13%, linoleic acid 42%, linolenic acid 3%, and other 5%.

[0028] The following test samples 1 to 3 were prepared or prepared as test samples used to evaluate the promotion of defensin expression described below.

[0029] Test sample 1 (glycerin solution of sodium lysophosphatidic acid) 2.5 g of the sodium lysophosphatidate-containing material obtained in Synthesis Example 1 and 7.5 g of glycerin were mixed and stirred at 70° C. for 3 hours to obtain a glycerin solution of sodium lysophosphatidate.

[0030] Test sample 2 (glycerin solution of aluminum lysophosphatidic acid) 2.5 g of the sodium lysophosphatidate-containing material obtained in Synthesis Example 1, 7.375 g of glycerin, and 0.125 g of aluminum hydroxide were mixed and stirred at 70° C. for 3 hours to obtain a glycerin solution of aluminum lysophosphatidate.

[0031] Test sample 3 (commercially available lysophosphatidic acid-containing product) A commercially available lysophosphatidic acid-containing product, PrimeLipid ALPA (a composition containing lysophosphatidic acid, lysolecithin, glycerin, water, and aluminum hydroxide; manufactured by Nippon Fine Chemical Industries Co., Ltd.), was used as is.

[0032] <Defensin expression promotion evaluation 1> Normal human epidermal keratinocytes were plated in a 24-well plate at 1.0 × 10 5 Cells were seeded at a concentration of 1000 cells / well and cultured in HuMedia-KG2 medium (Kurabo Industries, Ltd.) for 24 hours, then replaced with HuMedia-KB2 medium (Kurabo Industries, Ltd.). Each test sample was added to the medium at a concentration of 0.01% and cultured for an additional 4 hours. The gene expression levels of hBD-1 and hBD-3 (normalized by GAPDH) were then measured by real-time PCR. As a control, the gene expression levels in medium supplemented with glycerin as the test sample were also measured. The results are shown in Table 1 as relative values, with the average value of 3 wells taken as the control expression level of 100.

[0033] [Table 1]

[0034] <Defensin expression promotion evaluation 2> Normal human epidermal keratinocytes were plated in a 96-well plate at 1.5 × 10 4 Cells were seeded at a concentration of 1000 cells / well and cultured in HuMedia-KG2 medium (Kurabo Industries, Ltd.) for 24 hours, then replaced with HuMedia-KB2 medium (Kurabo Industries, Ltd.). Each test sample was added to the medium at a concentration of 0.01% and cultured for an additional 48 hours. The amount of hBD-3 in the culture supernatant was then quantified by ELISA. The amount of protein in each cultured cell was also quantified by BCA, and the amount of hBD-3 per unit protein was calculated. As a control, the amount of hBD-3 in medium supplemented with glycerol as the test sample was quantified in the same manner. The results are shown in Table 2, where the average value of four wells is used and the expression level of the control is set to 100.

[0035] [Table 2]

[0036] The results in Tables 1 and 2 indicate that lysophosphatidic acid or a salt thereof significantly increases the expression levels of hBD-1 and hBD-3. Therefore, it is believed that lysophosphatidic acid or a salt thereof can be used as an active ingredient in defensin expression promoters.

[0037] <Evaluation of acne bacteria growth inhibition> 3.0 x 10 normal human epidermal keratinocytes in a T75 flask for adherent cells 6 The cells were seeded at a concentration of 1000 cells / plate and cultured in HuMedia-KG2 medium (Kurabo Industries, Ltd.) for 24 hours, then replaced with HuMedia-KB2 medium (Kurabo Industries, Ltd.) and cultured for another 24 hours. Test sample 2 was then added to the medium to a concentration of 0.01%, and the culture was continued for an additional 48 hours, after which the culture supernatant was separated from the culture medium. As a control, the culture was carried out in the same manner except that glycerin was added instead of the test sample, and the culture supernatant was separated from the culture medium. 0.9 mL of the culture supernatant of the test sample or control taken above and 0.09 mL of GAM medium (10x) were added to a 1.5 mL microtube, and a 5.0 x 10 5 0.01 mL of a solution of Cutibacterium acnes (cfu / mL) was inoculated and cultured under anaerobic conditions at 37°C. The number of viable bacteria was measured by the colony formation method 0, 24, 48, and 72 hours after the start of culture. Based on the obtained numbers of viable bacteria in the test sample and the control, the rate of inhibition of acne bacteria growth of the test sample relative to the control was calculated using the following formula, and the results are shown in Table 3.

number

[0038] [Table 3]

[0039] The results in Table 3 demonstrate that lysophosphatidic acid or a salt thereof significantly inhibits the proliferation of Propionibacterium acnes by promoting the expression of hBD-3. Therefore, it is believed that lysophosphatidic acid or a salt thereof can be used as an active ingredient in an agent for inhibiting the proliferation of Propionibacterium acnes.

Claims

1. A defensin expression promoter comprising lysophosphatidic acid or a salt thereof.

2. The defensin expression promoter according to claim 1, wherein the defensin is human β-defensin-1 or human β-defensin-3.

3. A composition for improving antibacterial activity on the skin or in the body, comprising the defensin expression promoter according to claim 1 or 2.

4. An acne bacteria proliferation inhibitor containing lysophosphatidic acid or a salt thereof.

5. A composition for preventing or ameliorating acne, comprising the P. acnes growth inhibitor according to claim 4.

Images