Cyclic phosphatidic acid-containing composition and cosmetic method

Abstract

To provide a composition containing a single active ingredient that can bring about a plurality of cosmetic effects efficiently and conveniently, and a beauty method that can meet the consumer needs for many cosmetic effects efficiently and conveniently.SOLUTION: A cyclic phosphatidic acid sodium-containing composition is used alone and at a low concentration and exhibits a wide range of four actions simultaneously: collagen production promoting action, fibroblast growth promoting action, ceramide production promoting action, and hyaluronic acid production promoting action. There is also provided a food containing, as an active ingredient, cyclic phosphatidic acid sodium, having the actions of improving wrinkles and dry skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient for use in promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production.The present invention also relates to a non-therapeutic cosmetic method for promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production by administering a composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient to a subject. [Background technology]

[0002] In recent years, as various beauty needs have increased, various effects are being sought from beauty-related oral and topical preparations. Consumers choose oral and topical preparations depending on the beauty effect they desire, but when a wide range of beauty effects are desired, they have to purchase and use multiple oral and topical preparations, which is a hassle and costly problem. Therefore, there is a market need for oral and topical preparations that can exert multiple beauty effects independently.

[0003] When developing oral or topical formulations that can exert multiple beauty effects, it is common to select a single active ingredient for each desired beauty effect, resulting in the formulation of multiple active ingredients. However, incorporating multiple active ingredients into a single formulation poses challenges, including significant labor and cost in terms of raw material procurement and manufacturing, as well as challenges in formulation technology. Specifically, incorporating multiple active ingredients into a single formulation can easily disrupt the balance of pH and salt concentration, particularly in liquid formulations, resulting in poor stability over time. Even in solid formulations, unexpected interactions between the active ingredients can occur. Furthermore, this can lead to poor texture in oral formulations and unpleasant stickiness or sliminess in topical formulations. Furthermore, this can limit the formulation's flexibility by limiting the scope for incorporating ingredients other than the active ingredient.

[0004] Cyclic phosphatidic acid (cPA) is a known physiologically active lipid ubiquitously present in various organisms. While structurally similar to lysophosphatidic acid (LPA), cPA possesses a distinctive structure with cyclic phosphate groups at the sn-2 and sn-3 positions of the glycerol backbone (Non-Patent Document 1). This cyclic phosphate structure is known to be essential for the various physiological activities of cPA, including cytoskeleton construction and remodeling, inhibition of cancer cell invasion and metastasis, promotion of neuronal survival and differentiation, respiratory stimulation, and pain suppression (Non-Patent Document 2).

[0005] Furthermore, cyclic phosphatidic acid is known to have various effects on the skin, and is therefore used as a cosmetic ingredient in some cases (Non-Patent Document 2). However, there have not been many examples of cyclic phosphatidic acid being applied to cosmetics, and there are no known examples of its application to food. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Biochemistry, 90(6), 757-765, 2018 [Non-patent document 2] Bioscience and Industry, 69(1), 44-45, 2011 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a composition that can efficiently and simply provide multiple cosmetic effects by itself, and another object of the present invention is to provide a cosmetic method that can efficiently and simply meet the needs of consumers seeking multiple cosmetic effects. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have surprisingly found that cyclic sodium phosphatidate, even when used alone as an active ingredient at a low concentration, can simultaneously exhibit four broad effects, namely, collagen production promotion, fibroblast proliferation promotion, ceramide production promotion, and hyaluronic acid production promotion. Furthermore, they have found for the first time that foods containing cyclic sodium phosphatidate as an active ingredient have anti-wrinkle and anti-dry skin effects, which has led to the completion of the present invention.

[0009] That is, the present invention is as specified by the following items. (1) A composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient for use in promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production. (2) The composition according to (1) above, wherein the promotion of ceramide production is due to the promotion of expression of the sphingomyelin phosphodiesterase 1 (SMPD1) gene. (3) The composition according to (1) or (2) above, wherein the promotion of hyaluronic acid production is due to the promotion of expression of the hyaluronan synthase 3 (HAS3) gene. (4) The composition according to any one of (1) to (3) above, which is used for improving wrinkles and dry skin. (5) The composition according to (4) above, wherein the improvement of dry skin is due to the suppression of transepidermal water loss (TEWL). (6) The composition according to any one of (1) to (5) above, wherein the cyclic phosphatidic acid or a salt thereof is cyclic sodium phosphatidate. (7) The composition according to any one of (1) to (6) above, which contains 1.5 to 50 μg / mL of cyclic phosphatidic acid or a salt thereof as an active ingredient. (8) The composition according to any one of (1) to (7) above, which is an oral or external preparation. (9) The composition according to (8) above, wherein the oral or topical agent is a food product, cosmetic, quasi-drug, or pharmaceutical. (10) The composition according to (8) or (9) above, wherein the oral preparation is orally administered at least 3.6 mg of cyclic phosphatidic acid or a salt thereof as an active ingredient per day for at least 4 weeks. (11) A non-therapeutic cosmetic method for promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production by administering to a subject a composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient. (12) The non-therapeutic cosmetic method according to (11) above, further for improving wrinkles and dry skin. [Effects of the Invention]

[0010] According to the present invention, there is provided an excellent composition suitable for use as an internal or external preparation. Specifically, by incorporating cyclic phosphatidic acid or a salt thereof as an active ingredient, even when the active ingredient is used alone and at a low concentration, it is possible to simultaneously exert four broad effects, namely, collagen production promoting effect, fibroblast proliferation promoting effect, ceramide production promoting effect, and hyaluronic acid production promoting effect, and further to simultaneously exert wrinkle improving effect and dry skin improving effect, and the composition can be efficiently and simply prepared, which has good stability over time, good texture and feel when used, and high formulation flexibility.

[0011] Furthermore, the present invention can provide a cosmetic method that can efficiently and simply meet the needs of consumers seeking various cosmetic effects. Specifically, a non-therapeutic cosmetic method can be provided that can simultaneously obtain four broad effects, namely, collagen production promoting effect, fibroblast proliferation promoting effect, ceramide production promoting effect, and hyaluronic acid production promoting effect, by simply administering a composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient to a subject, and further, can simultaneously exhibit wrinkle improvement effect and dry skin improvement effect. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the results (average values) of I. Collagen production promotion test in Example 1. In the figure, "NcPA" represents a cyclic sodium phosphatidate-containing composition, "hyaluronic acid" represents a hyaluronic acid-containing composition, "proteoglycan" represents a proteoglycan-containing composition, "collagen" represents a collagen-containing composition, and "ceramide" represents a ceramide-containing composition. [Figure 2] This is a diagram showing the results (average values) of II. Fibroblast proliferation promotion test in Example 1. In the figure, "NcPA" represents a cyclic sodium phosphatidate-containing composition, "hyaluronic acid" represents a hyaluronic acid-containing composition, "proteoglycan" represents a proteoglycan-containing composition, "collagen" represents a collagen-containing composition, and "ceramide" represents a ceramide-containing composition. [Figure 3] 3 shows the results (average values) of III. Ceramide synthase gene expression promotion test in Example 1. In the figure, "NcPA" represents a cyclic sodium phosphatidate-containing composition, "hyaluronic acid" represents a hyaluronic acid-containing composition, "proteoglycan" represents a proteoglycan-containing composition, "collagen" represents a collagen-containing composition, and "ceramide" represents a ceramide-containing composition. [Figure 4] IV. A diagram showing the results (average values) of the hyaluronic acid synthase gene expression promotion test in Example 1. In the figure, "NcPA" represents a cyclic sodium phosphatidate-containing composition, "hyaluronic acid" represents a hyaluronic acid-containing composition, "proteoglycan" represents a proteoglycan-containing composition, "collagen" represents a collagen-containing composition, and "ceramide" represents a ceramide-containing composition. [Figure 5]Figure 5 shows the results of V. statistical analysis of Example 1. Specifically, the "1 / P" values ​​shown in Tables 9 to 12 are plotted as radar charts for the statistical analysis results for each test substance (test concentration 15.625 μg / mL) in the four test systems I. to IV. of Example 1. Note that the axes in Figure 5 are plotted on a logarithmic scale, and the value "20" on the axis is the reciprocal of P=0.05, so the area inside the value "20" on the axis is "no significant difference," and the area outside is "significant difference." In the figure, "NcPA" represents a composition containing cyclic sodium phosphatidate, "hyaluronic acid" represents a composition containing hyaluronic acid, "proteoglycan" represents a composition containing proteoglycan, "collagen" represents a composition containing collagen, and "ceramide" represents a composition containing ceramide. [Figure 6] FIG. 1 is a graph showing the results of I. Wrinkle improvement test in Example 2 (wrinkle score; mean value±SE). [Figure 7] FIG. 1 shows the results (reduction in wrinkles) of subject A in I. Wrinkle Improvement Test in Example 2. [Figure 8] FIG. 1 is a graph showing the results (reduction in wrinkles) of subject B in I. Wrinkle improvement test of Example 2. [Figure 9] FIG. 1 shows the results of II. Dry Skin Improvement Test (TEWL; mean±SE) in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0013] The composition of the present invention can be used for the following purposes (purposes of use): "to promote collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production."

[0014] In the present invention, "promotion of collagen production" refers to the promotion of collagen production in the body. "Promotion of collagen production" in the present invention can be evaluated by known methods, and is not particularly limited, but can be evaluated, for example, by measuring the amount of type I collagen produced. Collagen is a protein that mainly constitutes the dermis, ligaments, bones, cartilage, etc. of vertebrates, and is classified into types I to VII, each of which is known to have different properties and distribution locations. The "collagen" in the present invention may be any of types I to VII, but type I collagen, which is the main component of collagen fibers most abundant in the body, is particularly preferred. Increasing collagen in the body is known to be effective in improving skin elasticity, firmness, sagging, wrinkles, etc.

[0015] In the present invention, "promotion of fibroblast proliferation" means promoting the proliferation of fibroblasts in vivo. "Promotion of fibroblast proliferation" in the present invention can be evaluated by known methods, including, but not limited to, the MTT assay. Here, fibroblasts are mesoderm-derived cells scattered throughout connective tissues throughout the body. In the skin, fibroblasts reside in the dermis and perform metabolism, such as the synthesis and degradation of extracellular matrix in the dermis and basement membrane. This extracellular matrix is ​​known to be directly involved in skin elasticity, firmness, moisture, metabolism, and the like.

[0016] In the present invention, "promotion of ceramide production" refers to promotion of ceramide production in the body. "Promotion of ceramide production" in the present invention can be evaluated by known methods, including, but not limited to, measuring the amount of ceramide produced or the expression level of a gene involved in ceramide production. Examples of genes involved in ceramide production include the sphingomyelin phosphodiesterase 1 (SMPD1) gene, which is a ceramide synthase gene. SMPD1 is an enzyme involved in the synthesis of ceramide from sphingomyelin in the body. "Promotion of ceramide production" in the present invention may be due to promotion of SMPD1 gene expression. Ceramide is a type of sphingolipid that plays an important role in the moisturizing and barrier functions of the skin in the stratum corneum, the outermost layer of the skin. After being produced and secreted in epidermal cells, ceramide forms intercellular lamellae in the stratum corneum, exerting a skin moisturizing effect and providing skin protection and preventing rough skin.

[0017] In the present invention, "promotion of hyaluronic acid production" refers to the promotion of hyaluronic acid production in the body. "Promotion of hyaluronic acid production" in the present invention can be evaluated by known methods, including, but not limited to, measuring the amount of hyaluronic acid produced or the expression level of a gene involved in hyaluronic acid production. Examples of genes involved in hyaluronic acid production include the hyaluronan synthase 3 (HAS3) gene, which is a hyaluronic acid synthase gene. HAS3 is one of three isoforms of hyaluronic acid synthase present in mammals (HAS1, HAS2, and HAS3), and is an enzyme involved in hyaluronic acid synthesis primarily in the epidermis. "Promotion of hyaluronic acid production" in the present invention may be due to the promotion of HAS3 gene expression. Hyaluronic acid is a type of mucopolysaccharide present in the body. Its high water-retaining capacity is known to play an important role in improving skin moisturizing function, preventing wrinkles and sagging, and maintaining skin and articular cartilage function.

[0018] The composition of the present invention can also be used for the purpose of "improving wrinkles and dry skin."

[0019] "Wrinkle improvement" in the present invention means a reduction in the area and / or number of wrinkles in a certain area of ​​skin. "Wrinkle improvement" in the present invention can be evaluated by known methods, and is not particularly limited, and can be evaluated, for example, by visual inspection of the skin or image analysis. Here, wrinkles refer to groove-like lines that occur on the skin surface. The "wrinkles" in the present invention may be, for example, wrinkles caused by factors such as dryness wrinkles resulting from dryness on the skin surface, UV wrinkles caused by UV damage to the skin, and expression wrinkles caused by "skin folding marks" due to changes in facial expression, such as wrinkles at the corners of the eyes when smiling. In terms of location and shape, the "wrinkles" may be any of the following: linear wrinkles that occur at the corners of the eyes or between the eyebrows; figure wrinkles on the cheeks or neck where relatively deep grooves intersect with each other to form diamond or triangle shapes; and fine crepe wrinkles that occur on relaxed skin in the thighs, abdomen, etc. It is known that the formation of wrinkles is a complex combination of internal factors, such as the decline in the function of individual cells that make up the skin due to genetics and aging, and changes in systemic hormone levels, as well as external factors, such as UV exposure and the effects of dryness. In particular, UV exposure is thought to cause cell damage, cell apoptosis, a decrease in the proliferation activity of fibroblasts, the degeneration and destruction of collagen and elastin fibers, the accumulation of waste products due to a decrease in the vascular system, and a decrease in nutrient supply, resulting in a decrease in skin elasticity and leading to the formation of wrinkles.

[0020] In the present invention, "dry skin improvement" means that the decline in the skin's moisturizing function is inhibited or that the skin's moisturizing function is improved. "Dry skin improvement" in the present invention can be evaluated by known methods, including, but not limited to, measuring the moisture content of the stratum corneum or transepidermal water loss (TEWL). TEWL refers to the amount of moisture that volatilizes unconsciously from the body through the stratum corneum of the epidermis and is used as an indicator of the skin's barrier function, one of its important functions. "Dry skin improvement" in the present invention may be due to the suppression of TEWL. Furthermore, "dry skin" in the present invention refers to dry skin with a decline in moisturizing function. Normal skin has the ability to retain moisture (moisturizing function), and this moisturizing function allows the skin to maintain its flexibility and barrier function, thereby maintaining a good condition. On the other hand, when the skin's moisturizing function declines and the skin becomes dry, the skin's condition deteriorates, its texture becomes uneven, its flexibility decreases, and it becomes more susceptible to rough skin.

[0021] The composition of the present invention contains cyclic phosphatidic acid or a salt thereof as an active ingredient.

[0022] The "cyclic phosphatidic acid" in the present invention is not particularly limited as long as it is a lipid having a cyclic phosphate group structure. Specifically, the cyclic phosphatidic acid has cyclic phosphate groups at the sn-2 and sn-3 positions of the glycerol backbone. Note that "cyclic phosphatidic acid" is sometimes called "cyclic lysophosphatidic acid."

[0023] The "salt of cyclic phosphatidic acid" in the present invention may be a salt with a physiologically acceptable base or the like, and examples thereof include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; heavy metal salts such as zinc salt; ammonium salt; salts with basic amino acids or basic peptides, with sodium salts of cyclic sodium phosphatidate being particularly preferred. The method for producing cyclic sodium phosphatidate is not particularly limited, and examples thereof include the production method disclosed in JP 2016-183167 A.

[0024] The content of cyclic phosphatidic acid or a salt thereof, which is an active ingredient in the composition of the present invention, may be any amount that can exhibit collagen production promoting action, fibroblast proliferation promoting action, ceramide production promoting action, hyaluronic acid production promoting action, and further wrinkle improving action and dry skin improving action. For example, it may be 0.5 μg / mL or more, 1.0 μg / mL or more, 1.5 μg / mL or more, 3.0 μg / mL or more, 5.0 μg / mL or more, 6.0 μg / mL or more, 10 μg / mL or more, 20 μg / mL or more, 30 μg / mL or more, 40 μg / mL or more, 50 μg / mL or more, 60 μg / mL or more, 70 μg / mL or more, 80 μg / mL or more, 80 μg / mL or more, 90 μg / mL or more, 10 ... 0.0 μg / mL or more, 12.5 μg / mL or more, 25.0 μg / mL or more, 50.0 μg / mL or more, for example, 150.0 μg / mL or less, 125.0 μg / mL or less, 100.0 μg / mL or less, 75.0 μg / mL or less, 50.0 μg / mL or less, 25.0 μg / mL or less, 12.5 μg / mL or less, 10.0 μg / mL or less, 6.0 μg / mL or less, 5.0 μg / mL or less, 3.0 μg / mL, 1.5 μg / mL or less. That is, for example, in a composition containing 10% cyclic phosphatidic acid or a salt thereof as an active ingredient, the concentration is, for example, 5 μg / mL or more, 10 μg / mL or more, 15 μg / mL or more, 30 μg / mL or more, 50 μg / mL or more, 60 μg / mL or more, 100 μg / mL or more, 125 μg / mL or more, 250 μg / mL or more, 500 μg / mL or more, and, for example, 1500 μg / mL or less, 1250 μg / mL or less, 1000 μg / mL or less, 750 μg / mL or less, 500 μg / mL or less, 250 μg / mL or less, 125 μg / mL or less, 100 μg / mL or less, 60 μg / mL or less, 50 μg / mL or less, 30 μg / mL, 15 μg / mL or less.

[0025] The composition of the present invention can be used as an internal or external agent.

[0026] The subjects to which the oral or topical preparations are administered are not particularly limited as long as they are animals including humans, and are usually humans, but may also be animals other than humans (e.g., mammals such as mice, rats, hamsters, dogs, cats, sheep, goats, cows, pigs, and monkeys).

[0027] The "internal preparation" in the present invention is not particularly limited, and examples thereof include tablets (including sublingual tablets and orally disintegrating tablets), capsules (including soft capsules, hard capsules, and microcapsules), granules, powders, tablets, chewable preparations, troches, syrups, emulsions, suspensions, etc. The "internal preparation" in the present invention can also be referred to as an "oral preparation."

[0028] The "external preparation" in the present invention is not particularly limited, and examples thereof include liquid preparations (lotions, suspensions, emulsions, aerosols), ointments, creams, gels, patches, etc.

[0029] The oral preparation or topical preparation of the present invention can be used as a food, cosmetic, quasi-drug, or pharmaceutical. Specifically, the oral preparation of the present invention can usually be used as a food, quasi-drug, or pharmaceutical, and the topical preparation of the present invention can usually be used as a cosmetic, quasi-drug, or pharmaceutical.

[0030] The "food" in the present invention is not particularly limited as long as it is ingestible by the subject and is in an edible form, and examples thereof include solid, liquid, semi-liquid, granular, particulate, powder, capsule, cream, paste, etc. The "food" in the present invention also includes beverages.

[0031] Furthermore, the "food" in the present invention includes health foods, functional foods, foods for specified health uses, nutritional supplements, and foods for the sick. Health foods and the like may be in the form of ordinary foods, but are preferably in the form of supplements (tablets, granules, fine granules, tablets, chewable tablets, capsules (soft capsules, hard capsules), mini-bottled drinks, PET bottled drinks, etc.).

[0032] The "food" of the present invention may contain the active ingredient alone, but is typically formulated with other ingredients appropriate to the form of the food. The other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and examples include various proteins, sugars, fats, trace elements, vitamins, and organic acid salts such as citric acid and acetic acid. Depending on the type of food, the food of the present invention may also contain additives that are tolerated and commonly used in foods, such as sweeteners such as aspartame and stevia, acidulants such as citric acid, malic acid, and tartaric acid, and excipients such as dextrin and starch, as well as colorants, flavorings, bittering agents, buffers, thickening agents, gelling agents, stabilizers, gum bases, binders, diluents, emulsifiers, dispersants, suspending agents, antioxidants, preservatives, antiseptics, fungicides, color formers, bleaching agents, brighteners, enzymes, seasonings, and spice extracts.

[0033] Specific examples of "foods" in the present invention include, but are not limited to, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and dairy drinks (including concentrated concentrates and powders for adjusting these beverages); carbohydrate-containing foods and beverages such as rice, noodles, bread, and pasta; Western sweets such as cookies and cakes, Japanese sweets such as buns and yokan, candies, gum, and frozen and frozen desserts such as yogurt, pudding, and jelly; processed seafood and livestock foods such as kamaboko, chikuwa, hamburger steak, ham, and sausage; dairy products such as processed milk, fermented milk, yogurt, butter, and cheese; oils and fats and processed oil-and-fat foods such as margarine, mayonnaise, shortening, whipped cream, and dressing; and seasonings such as sauces and dressings.

[0034] The "cosmetics" of the present invention are not particularly limited, and examples thereof include lotions, emulsions, creams, beauty serums, hair tonics, hair growth agents, facial masks, lipsticks, lip balms, makeup base lotions, makeup base creams, foundations, eye colors, cheek colors, shampoos, conditioners, hair liquids, hair tonics, permanent wave agents, hair colors, treatments, bath additives, hand creams, leg creams, neck creams, and body lotions.

[0035] The "cosmetics" of the present invention may contain an active ingredient alone, but are usually formulated with other ingredients appropriate to the form of the cosmetic. The other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and examples include water (purified water, hot spring water, deep sea water, etc.), alcohols, oils, surfactants, metal soaps, gelling agents, powders, water-soluble polymers, film-forming agents, resins, UV protection agents, clathrate compounds, antibacterial agents, fragrances, deodorants, salts, pH adjusters, cooling agents, animal- or microbial-derived extracts, plant extracts, blood circulation promoters, astringents, antiseborrheic agents, anti-inflammatory agents, cell activators, moisturizers, chelating agents, keratolytic agents, enzymes, hormones, vitamins, and the like, all of which are commonly used in cosmetic formulations.

[0036] The "quasi-drugs" and "drugs" of the present invention are not particularly limited, and examples thereof include tablets (including sugar-coated tablets), capsules, granules, powders, pills, oral solutions, suspensions, emulsions, syrups, ointments, gels, external powders, sprays, inhalable powders, injections (solutions, suspensions, emulsions, solid preparations to be dissolved when used, etc.), drip infusions, suppositories, etc.

[0037] The "quasi-drugs" and "drugs" of the present invention may contain an active ingredient alone, but are usually formulated together with other pharmaceutically acceptable ingredients appropriate to the form of each drug. Here, the other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and examples thereof include carriers, excipients, diluents, binders, lubricants, disintegrants or disintegration aids, solubilizers, stabilizers, preservatives, antiseptics, bulking agents, thickeners, emulsifiers, dispersants, suspending agents, buffers, etc.

[0038] The oral preparation of the present invention may be taken in a dosage and administration method that can achieve the anti-wrinkle and anti-dry skin effects, but it is preferred that cyclic phosphatidic acid or its salt be orally taken as an active ingredient in an amount of at least 3.6 mg per day for at least 4 weeks in an adult. The number of times of daily intake is not particularly limited, and examples include taking at least 3.6 mg of the active ingredient in a single dose per day, taking at least 3.6 mg of the active ingredient in two divided doses per day, or taking at least 3.6 mg of the active ingredient in three or more divided doses per day.

[0039] The present invention also provides a non-therapeutic cosmetic method by administering to a subject a composition containing cyclic phosphatidic acid or a salt thereof as an active ingredient. The composition of the present invention can be used for the purposes of "promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production" and further "alleviating wrinkles and dry skin."

[0040] Here, the "composition" refers to an oral or topical agent, and the oral or topical agent may be a food, cosmetic, quasi-drug, or pharmaceutical. The "subject" is not particularly limited, but is preferably a subject who desires collagen production promotion, fibroblast proliferation promotion, ceramide production promotion, and hyaluronic acid production promotion, as well as wrinkle and dry skin improvement. Here, "administration" is preferably oral administration if the composition is an oral agent, and particularly preferably oral administration of at least 3.6 mg of cyclic phosphatidic acid or a salt thereof per day for at least 4 weeks to an adult. If the composition is an external agent, application to the skin surface is preferred. Here, the skin surface to which the composition is applied is not particularly limited, and examples include the face, such as the forehead, eyes, corners of the eyes, cheeks, chin, nose and surrounding areas, lips, and mouth; the neck and surrounding areas; the hands and wrists, such as the fingers, backs, and palms; the toes, tops of the feet, soles, heels, ankles; elbows, arms, legs, chest, chest area, abdomen, and back. Preferably, the area is the face, the neck and surrounding areas, or the hands, including the fingers and back of the hand, and more preferably the face.

[0041] Furthermore, the term "non-therapeutic cosmetic method" in the present invention means a cosmetic method, not a therapeutic method. [Example]

[0042] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0043] <Cell test> I. Collagen production promotion test 1. Test item Test substance: The following five commercially available food ingredients were used. (1) Cyclic sodium phosphatidate-containing composition Cyclic sodium phosphatidate (active ingredient) content: 10% The product used was "NcPA (registered trademark) 10P" (manufactured by SANSHO Co., Ltd.), manufactured by the manufacturing method disclosed in JP 2016-183167 A. (2) Hyaluronic acid-containing composition Hyaluronic acid (active ingredient) content standard: 90-100% (3) Proteoglycan-containing composition Proteoglycan (active ingredient) content standard: 20% or more (4) Collagen-containing composition Collagen (active ingredient) content standard (as protein): 90% or more (5) Ceramide-containing composition Glucosylceramide (active ingredient) content standard: 4% or more Positive control: (6) Ascorbyl phosphate magnesium salt (reagent)

[0044] 2. Test Method (1) Normal human dermal fibroblasts (Promocell) were cultured in a culture medium (D-MEM medium containing 10% FBS, Fujifilm Wako Pure Chemical Industries, Ltd.) to transition to the logarithmic growth phase while simultaneously ensuring the required number of cells. (2) Cells were seeded onto a test plate (96 wells) (seeding conditions: 2 × 10 3 cells / 100μL / well). (3) The cells were pre-cultured for 72 hours under conditions of 37°C and 5% CO2. (4) After pre-incubation, the culture medium was removed, and the test substance or positive control dissolved in the culture medium to each test concentration (15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL) was added to each well, followed by incubation for 24 hours at 37°C and 5% CO. Note that, for example, since the sodium cyclic phosphatidate-containing composition used as the test substance contains 10% cyclic sodium phosphatidate as the active ingredient as described above, the content of sodium cyclic phosphatidate (active ingredient) at each test concentration was 1.5625 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25.0 μg / mL, and 50.0 μg / mL, respectively. (5) The culture medium was collected from each well, and the amount of collagen produced in the culture medium was measured using a type I collagen measurement ELISA (human collagen type I kit, manufactured by A-Cell). The collagen production rate for each test sample group was calculated, with the amount of collagen produced in the control group (without test sample added) set at 100%. The test was performed five times, and the average and SD (standard deviation) were calculated (n = 5).

[0045] 3.Results The results are shown in Tables 1 and 2 and Figure 1. The composition containing cyclic sodium phosphatidate showed a high collagen production rate, demonstrating that the composition containing cyclic sodium phosphatidate has a high collagen production-promoting effect.

[0046] [Table 1] The values ​​in the table represent the collagen production rate (mean and SD; n=5) for each test substance at each test concentration.

[0047] [Table 2] The values ​​in the table represent the collagen production rate (mean and SD; n=5) of the positive control at each test concentration.

[0048] II. Fibroblast proliferation promotion test 1. Test item The same test substances and positive controls as those in (1) to (6) of "I. Collagen production promotion test" above were used.

[0049] 2. Test Method (1) Normal human dermal fibroblasts (Promocell) were cultured in a culture medium (D-MEM medium containing 10% FBS, Fujifilm Wako Pure Chemical Industries, Ltd.) to transition to the logarithmic growth phase while simultaneously ensuring the required number of cells. (2) Cells were seeded onto a test plate (96 wells) (seeding conditions: 2 × 10 3 cells / 100μL / well). (3) The cells were pre-cultured for 24 hours under conditions of 37°C and 5% CO2. (4) After pre-incubation, the culture medium was removed, and the test substance or positive control dissolved in culture medium to each test concentration (15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL) was added to each well, and the cells were cultured for 48 hours at 37°C under 5% CO2. (5) After 48 hours, the culture supernatant was removed, and 100 μL of D-MEM medium (Fujifilm Wako Pure Chemical Industries) containing 1 mg / mL MMT (Dojindo Laboratories) was added to each well, followed by culturing for 3 hours. (6) After 3 hours of culture, the MMT-containing D-MEM medium was removed, and 100 μL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, followed by washing twice. (7) 100 μL of 2-propanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and shaken to extract MMT. (8) The absorbance at 570 nm was measured, and the fibroblast proliferation rate for each test sample group was calculated, with the absorbance of the control group set at 100%. The test was performed five times, and the mean and SD (standard deviation) were calculated (n=5).

[0050] 3.Results The results are shown in Tables 3 and 4 and Figure 2. The composition containing cyclic sodium phosphatidate was found to have a fibroblast proliferation promoting effect.

[0051] [Table 3] The values ​​in the table represent the fibroblast proliferation rate (mean and SD; n=5) for each test substance at each test concentration.

[0052] [Table 4] The values ​​in the table represent the fibroblast proliferation rate (mean and SD; n=5) of the positive control at each test concentration.

[0053] III. Ceramide synthase gene expression promotion test 1. Test item Test substance: The same test substances as those in (1) to (5) of "I. Collagen production promotion test" above were used. Positive control: (7) Licorice extract Licorice powder, as specified in the Japanese Pharmacopoeia, was dissolved in 50% ethanol to a concentration of 1% glycyrrhizinic acid.

[0054] 2. Test Method (1) Normal human epidermal keratinocytes (Promocell) were cultured in a culture medium (D-MEM medium containing 10% FBS, Fujifilm Wako Pure Chemical Industries, Ltd.) to transition to the logarithmic growth phase while simultaneously ensuring the required number of cells. (2) Cells were seeded onto a test plate (12 wells) (seeding conditions: 2 × 10 3 cells / 100μL / well). (3) The cells were pre-cultured for 24 hours under conditions of 37°C and 5% CO2. (4) After pre-incubation, the culture medium was removed, and the test substance or positive control dissolved in culture medium to each test concentration (15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL) was added to each well, and the cells were cultured for 48 hours at 37°C under 5% CO2. (5) After the incubation, the cells were thoroughly washed with PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.), and RNA was extracted using NucleoSpin (registered trademark) RNA (Takara Bio Inc.). (6) Dilute a portion of the obtained RNA sample 50-fold and measure the absorbance at 260 nm (OD 260 ) was measured, and the total RNA amount was calculated using the following formula. Calculation formula: OD 260 ×40 (concentration coefficient; factor) × 50 (dilution rate) = Total RNA amount (ng / μL) (7) cDNA was prepared by PCR reverse transcription (37°C for 15 minutes, 85°C for 5 seconds) using PrimeScript® RT Master Mix (Takara Bio Inc.). For real-time PCR, SYBR® Premix Ex Taq™ II (Takara Bio Inc.) was used. 12.5 μL of SYBR® Premix Ex Taq™ II (TliRNase H Plus) (2X), 1 μL of PCR Forward Primer (10 μM), 1 μL of PCR Reverse Primer (10 μM), and 1 μL or 2 μL of cDNA were added, and the total volume was adjusted to 25 μL using RNase-free HO. The real-time PCR reaction consisted of an initial denaturation at 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60-65°C for 30 seconds, followed by one cycle of 60-95°C at 0.2 deg / s. This real-time PCR was used to quantify the expression of the sphingomyelin phosphodiesterase 1 (SMPD1) gene, a ceramide synthase gene, and the GAPDH gene was used as a housekeeping gene. (8) After the PCR reaction, the results were analyzed using the ΔΔCt method. The expression level in the control group (without test product) was set at 100%, and the expression rate in each test product group was calculated. The test was performed five times, and the average value and SD (standard deviation) were calculated (n = 5).

[0055] 3.Results The results are shown in Tables 5 and 6 and Figure 3. The composition containing cyclic sodium phosphatidate showed a particularly high SMPD1 gene expression rate compared to the other test substances. Therefore, it was confirmed that the composition containing cyclic sodium phosphatidate has a high SMPD1 gene expression promoting effect.

[0056] [Table 5] The values ​​in the table represent the SMPD1 gene expression rate (mean and SD; n=5) for each test substance at each test concentration.

[0057] [Table 6] The values ​​in the table represent the SMPD1 gene expression rate (mean and SD; n=5) for each test concentration of the positive control.

[0058] IV. Hyaluronic acid synthase gene expression promotion test 1. Test item Test substance: The same test substances as those in (1) to (5) of "I. Collagen production promotion test" above were used. Positive control: (8) N-acetylglucosamine (reagent)

[0059] 2. Test Method (1) Normal human dermal fibroblasts (Promocell) were cultured in a culture medium (D-MEM medium containing 10% FBS, Fujifilm Wako Pure Chemical Industries, Ltd.) to transition to the logarithmic growth phase while simultaneously ensuring the required number of cells. (2) Cells were seeded onto a test plate (96 wells) (seeding conditions: 2 × 10 3 cells / 100μL / well). (3) The cells were pre-cultured for 24 hours under conditions of 37°C and 5% CO2. (4) After pre-incubation, the culture medium was removed, and the test substance or positive control dissolved in culture medium to each test concentration (15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL) was added to each well, and the cells were cultured for 48 hours at 37°C under 5% CO2. (5) After the incubation, the cells were thoroughly washed with PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.), and RNA was extracted using NucleoSpin (registered trademark) RNA (Takara Bio Inc.). (6) Dilute a portion of the obtained RNA sample 50-fold and measure the absorbance at 260 nm (OD 260 ) was measured, and the total RNA amount was calculated using the following formula. Calculation formula: OD 260 ×40 (concentration coefficient; factor) × 50 (dilution rate) = Total RNA amount (ng / μL) (7) cDNA was prepared by PCR reverse transcription (37°C for 15 minutes, 85°C for 5 seconds) using PrimeScript® RT Master Mix (Takara Bio Inc.). For real-time PCR, SYBR® Premix Ex Taq™ II (Takara Bio Inc.) was used. 12.5 μL of SYBR® Premix Ex Taq™ II (TliRNase H Plus) (2X), 1 μL of PCR Forward Primer (10 μM), 1 μL of PCR Reverse Primer (10 μM), and 1 μL or 2 μL of cDNA were added, and the total volume was adjusted to 25 μL using RNase-free HO. The real-time PCR reaction consisted of an initial denaturation at 95°C for 10 seconds, followed by 45 cycles of 95°C for 5 seconds and 60-65°C for 30 seconds, followed by one cycle of 60-95°C at 0.2 deg / s. This real-time PCR was used to quantify the expression of the hyaluronan synthase 3 (HAS3) gene as a hyaluronan synthase gene, and the GAPDH gene was used as a housekeeping gene. (8) After the PCR reaction, the results were analyzed using the ΔΔCt method. The expression level in the control group (without test product) was set at 100%, and the expression rate in each test product group was calculated. The test was performed five times, and the average value and SD (standard deviation) were calculated (n = 5).

[0060] 3.Results The results are shown in Tables 7 and 8 and Figure 4. The composition containing cyclic sodium phosphatidate showed a high HAS3 gene expression rate, demonstrating that the composition containing cyclic sodium phosphatidate has a high HAS3 gene expression-promoting effect.

[0061] [Table 7] The values ​​in the table represent the HAS3 gene expression rate (mean and SD; n=5) for each test substance at each test concentration.

[0062] [Table 8] The values ​​in the table represent the HAS3 gene expression rate (mean and SD; n=5) for each test concentration of the positive control.

[0063] V. Statistical analysis 1. Method The test results from the four test systems I to IV above were used as the data to be analyzed, and the nonparametric multiple comparison test Steel-Dwass method (BellCurve for Excel software (ver. 3.209) Social Survey Research Information Co., Ltd.) was used for analysis. Specifically, the mean values ​​± SD (collagen production rate, fibroblast proliferation rate, SMPD1 gene expression rate, and HAS3 gene expression rate) at each test concentration of the five test substances in the test systems I to IV above were compared with the values ​​of the control group to which the test product had not been added. A P value of <0.05 was considered to be "significantly different."

[0064] 2.Results It was revealed that the cyclic sodium phosphatidate-containing composition exhibited significant effects compared to the control group at all test concentrations in all four test systems (I. to IV.). In particular, the analytical results for the lowest test concentration, 15.625 μg / mL, are shown in Tables 9 to 12 and Figure 5. Figure 5 is a radar chart showing the reciprocal of the P value, i.e., the "1 / P" value shown in Tables 9 to 12, for the statistical analysis results for each test substance (test concentration 15.625 μg / mL) in the four test systems (I. to IV.). The axis in Figure 5 is plotted on a logarithmic scale, and the value "20" on the axis is the reciprocal of P=0.05. Therefore, the area inside the value "20" on the axis represents "no significant difference," while the area outside the value "20" represents "significant difference."

[0065] As is clear from Tables 9-12 and Figure 5, of the five test substances, only the cyclic sodium phosphatidate-containing composition showed a significant effect compared to the control group at a test concentration of 15.625 μg / mL in all four test systems I to IV. The active ingredients (hyaluronic acid, proteoglycan, collagen, and ceramide) of the four test substances used in this study, except for the cyclic sodium phosphatidate-containing composition, are well known to have some degree of activity on skin tissue. However, in this analysis, these four test substances showed varying degrees of effect among the test systems (Figure 5). Therefore, the broad and balanced effects of the cyclic sodium phosphatidate-containing composition in promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production, especially at low concentrations, were found to be unique to the cyclic sodium phosphatidate-containing composition, which contains cyclic sodium phosphatidate as the active ingredient.

[0066] [Table 9] I. Statistical analysis of collagen production rate (n=5, *P<0.05, **P<0.01)

[0067] [Table 10] II. Statistical analysis of fibroblast proliferation rate (n=5, *P<0.05, **P<0.01)

[0068] [Table 11] III. Statistical analysis of sphingomyelin phosphodiesterase 1 (SMPD1) gene expression (n=5, *P<0.05, **P<0.01)

[0069] [Table 12] IV. Statistical analysis of hyaluronic acid synthase 3 (HAS3) gene expression (n=5, *P<0.05, **P<0.01) [Example]

[0070] <Human trials> I. Wrinkle improvement test 1. Test conditions Test substance: Enteric-coated hard capsule containing cyclic sodium phosphatidate Cyclic sodium phosphatidate (active ingredient) content: 1.8 mg / tablet The above active ingredient is derived from the product "NcPA (registered trademark) 10P" (manufactured by SANSHO Co., Ltd.), which is manufactured by the manufacturing method disclosed in JP 2016-183167 A. Subjects: 12 healthy adult men and women (20s to 50s) Test period: 28 days Number of evaluations: 2 times (day 0, day 28) Evaluation area: cheeks (left and right) All measurements were performed after washing the face and allowing the skin to acclimate for 15 minutes at constant temperature and humidity (22-23°C, RH 40-50%). 2. Test Method (1) Using a facial image analyzer (product name: VISIA® Evolution, manufactured by Canfield Scientific), facial images of the subjects were taken on the first day of the study (day 0), and the wrinkle score was evaluated from the captured images. The wrinkle score is a value that represents the proportion of wrinkles detected as areas of a certain length and shape (linear) within the analysis frame, and a lower wrinkle score indicates less wrinkled skin. (2) During the test period (days 1 to 28), subjects orally ingested two tablets of the test substance (active ingredient amount: 3.6 mg) per day. (3) On the 28th day, the subjects' faces were photographed again in the same manner as on day 0, and the wrinkle scores were evaluated from the photographed images. (4) The facial images and wrinkle scores of each subject were compared and analyzed on days 0 and 28. The mean values ​​and standard errors (SE) of the wrinkle scores for all subjects on days 0 and 28 were calculated (n = 12). 3.Results The analysis results of the wrinkle scores are shown in Table 13 and Figure 6. The average wrinkle score was significantly reduced on day 28 compared to day 0. As examples of representative subjects, facial images (left cheek) of subject A (female in her 50s) are shown in Figure 7 and subject B (female in her 40s) are shown in Figure 8. In both subjects, facial wrinkles (areas indicated by lines) were observed to be reduced on day 28 compared to day 0. Therefore, oral ingestion of the test substance containing cyclic sodium phosphatidate as the active ingredient was confirmed to have an effect of improving wrinkles.

[0071] [Table 13]

[0072] II. Dry skin improvement test 1. Test conditions Test substance: The same test substance as in "I. Wrinkle Improvement Test" above was used. Subjects: 7 healthy adult men and women (20s to 50s) Test period: 28 days Number of measurements: 2 times (day 0, day 28) Measurement area: cheeks (left and right) All measurements were performed after washing the face and allowing the skin to acclimate for 15 minutes at constant temperature and humidity (22-23°C, RH 40-50%). 2. Test Method (1) Using a transepidermal water loss meter (product name: Tewameter (registered trademark) TM300, manufactured by Courage + Khazaka Electronics), the subjects' transepidermal water loss (TEWL) was measured on the first day of the study (day 0). Note that a lower TEWL value indicates a higher level of skin barrier function. (2) During the test period (days 1 to 28), subjects orally ingested two tablets of the test substance (active ingredient amount: 3.6 mg) per day. (3) On the 28th day, the subjects' TEWL was measured again in the same manner as on the 0th day. (4) The mean values ​​and SE (standard error) of TEWL for all subjects on days 0 and 28 were calculated and compared (n=7). 3.Results The TEWL analysis results are shown in Table 14 and Figure 9. The mean TEWL value was significantly reduced on day 28 compared to day 0. Therefore, oral ingestion of the test substance containing cyclic sodium phosphatidate as an active ingredient was confirmed to have a TEWL-suppressing effect.

[0073] [Table 14]

[0074] III. Summary In addition to the above-mentioned wrinkle and dry skin improvement, subjects in human tests also reported effects such as "finer skin texture," "smoother skin," and "younger skin." As shown in Example 1, the composition containing cyclic sodium phosphatidate has a wide and balanced effect in at least promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production. It is believed that these effects contribute to wrinkle improvement and dry skin improvement, as well as finer skin texture, smoother skin, and more youthful skin. From these facts, it can be said that cyclic sodium phosphatidate is a very suitable active ingredient for efficiently achieving many beauty effects. [Industrial Applicability]

[0075] The present invention can be used in the fields of manufacturing internal and external preparations (foods, cosmetics, quasi-drugs or pharmaceuticals) for the purposes of promoting collagen production, fibroblast proliferation, ceramide production, and hyaluronic acid production, as well as improving wrinkles and dry skin.

Claims

1. An oral preparation containing cyclic phosphatidic acid or a salt thereof as an active ingredient for use in promoting collagen production, promoting fibroblast proliferation, promoting ceramide production, promoting hyaluronic acid production, improving wrinkles, and improving dry skin, The oral preparation, wherein at least 3.6 mg of cyclic phosphatidic acid or a salt thereof is orally administered per day for at least 4 weeks.

2. 2. The oral preparation according to claim 1, wherein the promotion of ceramide production is due to the promotion of expression of the sphingomyelin phosphodiesterase 1 (SMPD1) gene.

3. 3. The oral preparation according to claim 1 or 2, wherein the promotion of hyaluronic acid production is due to the promotion of expression of the hyaluronan synthase 3 (HAS3) gene.

4. 4. The oral preparation according to claim 1, wherein the improvement of dry skin is due to the suppression of transepidermal water loss (TEWL).

5. The oral preparation according to any one of claims 1 to 4, wherein the cyclic phosphatidic acid or a salt thereof is cyclic sodium phosphatidate.

6. The internal preparation according to any one of claims 1 to 5, which is a food product, a quasi-drug, or a pharmaceutical product.

7. A non-therapeutic cosmetic method (excluding medical procedures on humans) for promoting collagen production, fibroblast proliferation, ceramide production, hyaluronic acid production, wrinkle improvement, and dry skin improvement, comprising having a subject orally ingest at least 3.6 mg of cyclic phosphatidic acid or a salt thereof per day for at least four weeks using an oral preparation containing cyclic phosphatidic acid or a salt thereof as an active ingredient.

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