Ceramide composition

Abstract

Provided is a novel ceramide composition. Specifically, provided is a ceramide composition extracted from a chicken leg by means of ethanol.

Description

[Technical Field]

[0001] This disclosure relates to ceramide compositions and their uses. All references cited herein are incorporated herein by reference. [Background technology]

[0002] In recent years, ceramides have been expanding their market as functional cosmetic ingredients, as they are the main component of intercellular lipids in human epidermis. It is known that keratin ceramides gradually decrease with age, and therefore, supplementing with topical ceramides is thought to be important for maintaining healthy skin. Furthermore, since ceramides present as intercellular lipids in the stratum corneum are free ceramides, free ceramides are also attracting attention.

[0003] To date, research has also been conducted on how to efficiently prepare useful ceramides (for example, Patent Documents 1 and 2). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2012-126910 [Patent Document 2] International Publication No. 2019 / 049964 [Overview of the project] [Problems that the invention aims to solve]

[0005] The inventors of this invention conducted research with the aim of discovering new functions of ceramide. [Means for solving the problem]

[0006] The inventors have discovered that certain bio-derived ceramide compositions may be able to perform specific functions particularly efficiently. Furthermore, they have found that among bio-derived ceramide compositions, a ceramide-containing extract obtained by extracting chicken feet with ethanol (chicken foot ethanol-extracted ceramide composition) can exert a particularly desirable effect of enhancing epithelial barrier function. Based on these findings, further improvements have been made. This disclosure includes, for example, the following subjects: Section 1. Chicken foot ethanol extract ceramide composition. Section 2. The composition according to item 1, comprising ω-O-acylceramide. Section 3. The composition according to item 2, wherein the contained ω-O-acylceramide is of the EOS type and the terminal acyl group is a linoleic acid group. Section 4. The composition according to item 2 or 3, wherein 0.1 to 2% by mass of the ceramide contained is ω-O-acylceramide. Section 5. A composition according to any one of items 1 to 4, for strengthening the epithelial barrier. Section 6. A composition according to any one of items 1 to 5, for strengthening the skin barrier or the intestinal barrier. Section 7. A skin barrier strengthening composition as described in item 6, for caring for skin damage caused by ultraviolet rays. Section 8. The intestinal barrier strengthening composition according to item 6, for strengthening tight junctions of intestinal epithelial cells. Section 9. The composition according to any one of claims 1 to 8, wherein 90% by mass or more of the contained ceramides are free ceramides whose ceramide skeleton is a combination of (a sphingoid base having two hydroxyl groups, 18 carbon atoms, and one carbon-carbon double bond)-(a fatty acid having 16 to 34 carbon atoms, 0 or 1 carbon-carbon double bond, and 0 hydroxyl groups). Section 10. The composition according to any one of items 1 to 8, wherein 90% by mass or more of the ceramides contained are ceramide NS. [Effect of the Invention]

[0007] A novel bio-derived ceramide composition is provided. In particular, a ceramide-containing extract obtained by extracting from chicken legs with ethanol (chicken leg ethanol extract ceramide composition) is provided. The said ceramide composition can be preferably used particularly for strengthening the epithelial barrier. [Brief Description of the Drawings]

[0008] [Figure 1] The results of measuring the number, area, and membrane potential activity of mitochondria using an IN Cell Analyzer 2200 after adding ceramide to HaCaT cells and culturing for 48 hours are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 2] The results of measuring ROS using an IN Cell Analyzer 2200 after adding ceramide to HaCaT cells irradiated with 10 mJ / cm2 of UVB and culturing for 24 hours are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 3] The results of measuring the number, area, and membrane potential activity of mitochondria using an IN Cell Analyzer 2200 after adding ceramide to HaCaT cells irradiated with 10 mJ / cm2 of UVB and culturing for 24 hours are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 4] The results of analyzing the fatty acid composition of ceramide in HaCaT cells using LC-MS after adding ceramide to HaCaT cells and culturing for 48 hours are shown. [Figure 5]The supernatant of Caco-2 cells treated with ceramide for 24 hours was added to HaCaT cells irradiated with 10 mJ / cm2 of UVB. After 48 hours, the results of measuring the number, area, and membrane potential activity of mitochondria using an IN Cell Analyzer 2200 are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, # = 0.08 Student’s t-test) [Figure 6] The supernatant of Caco-2 cells treated with ceramide for 24 hours was added to HaCaT cells irradiated with 10 mJ / cm2 of UVB. After 48 hours, the results of measuring ROS using an IN Cell Analyzer 2200 are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, #p < 0.15, Student’s t-test) [Figure 7] The supernatant of Caco-2 cells treated with ceramide for 24 hours was added to HaCaT cells irradiated with 10 mJ / cm2 of UVB. After 48 hours, the results of measuring the number, area, and membrane potential activity of mitochondria using an IN Cell Analyzer 2200 are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 8] The results of measuring the mRNA expression level of Claudin-3 after adding ceramide to Caco-2 cells and culturing for 24 hours are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 9] The content ratio (mass%) of various free ceramide species in the soy sauce residue extract ceramide composition is shown.

Modes for Carrying Out the Invention

[0009] Hereinafter, each embodiment included in the present disclosure will be described in more detail. The present disclosure preferably includes, but is not limited to, specific bio-derived ceramide compositions and their uses, etc. The present disclosure includes all that is disclosed herein and can be recognized by those skilled in the art.

[0010] The ceramide compositions included in this disclosure are bio-derived ceramide compositions, and more specifically, soy sauce lees extract ceramide compositions, Aspergillus oryzae extract ceramide compositions, or chicken foot extract ceramide compositions. The term "ceramide composition" here refers to any composition containing ceramide. These bio-derived ceramides may be referred to as the ceramide compositions of this disclosure. Among the ceramide compositions of this disclosure, chicken foot extract ceramide compositions are preferred, and among the chicken foot extract ceramide compositions, ceramide-containing extracts obtained by extracting from chicken feet with ethanol (chicken foot ethanol extract ceramide compositions) are particularly preferred. Furthermore, the toe portion (momiji) of the chicken foot is particularly preferred. It is also preferable to use the epidermis portion of the chicken foot (especially the momiji) for ethanol extraction. The chicken feet used for ethanol extraction are preferably pre-dried, and particularly preferably in the form of a dried powder.

[0011] The ceramide compositions disclosed herein, particularly the chicken foot ethanol-extracted ceramide compositions, preferably exhibit the effect of enhancing epithelial barrier function. For this reason, they can be preferably used for strengthening epithelial barriers. Particularly preferred epithelial barriers include skin barriers (epidermal barriers) and intestinal barriers (intestinal epithelium).

[0012] The ceramide compositions of this disclosure, particularly the chicken foot ethanol extract ceramide composition, can be used to strengthen the skin barrier, and are especially preferred for preventing the effects of ultraviolet radiation on the skin (caring for damage). While we do not wish to be bound by theory, this is because applying the ceramide compositions of this disclosure to the skin may make it possible to restore mitochondria (particularly in number, area, or membrane potential activity) in skin cells that are damaged or have been damaged by ultraviolet radiation. They can be used preventively for skin that is damaged by ultraviolet radiation, or therapeutically for skin that has been damaged by ultraviolet radiation.

[0013] When the ceramide composition of this disclosure is used for skin (for strengthening the skin barrier), it can be used, for example, as a topical composition or an oral composition (for example, an oral pharmaceutical composition or a food composition), and it is preferable to use it as a topical composition.

[0014] Among the ceramide compositions disclosed herein, the chicken foot ethanol extract ceramide composition can be preferably used for the intestinal barrier, and is particularly preferably used for strengthening tight junctions in intestinal epithelial cells. While we do not wish to be bound by theory, this is because, by applying the chicken foot ethanol extract ceramide composition to the intestinal tract, it may be possible to enhance the expression of claudin (especially claudin 3) in intestinal cells. Furthermore, by strengthening tight junctions, the defense function against pathogens in the intestinal tract may also be enhanced. When used for such enhancement of infection defense, it can be used for infection prevention or for treatment after infection. Claudin is a major protein involved in the formation of tight junctions and plays a central role in the intercellular barrier at tight junctions.

[0015] Furthermore, the density and bonding properties of tight junctions can be measured using trans-epithelial electrical resistance (TER) as an indicator, and the ceramide composition of this disclosure can preferably improve the TER value.

[0016] Furthermore, when the ceramide composition of this disclosure is used in the intestinal tract (as an intestinal barrier), it can be used, for example, as an oral composition (e.g., an oral pharmaceutical composition or a food composition) and as a composition for intestinal administration. When used as a composition for intestinal administration, it can be administered directly to the intestines by surgical procedure, or it can be administered rectally.

[0017] Ceramides are compounds having a structure (-NH-CO-) in which the amino group (-NH2) of a sphingoid base is bonded to the carboxyl group (-COOH) of a fatty acid. Further bonding of polar groups such as sugars and phosphoric acid to the alcoholic hydroxyl group (-OH) of the sphingoid base of ceramide results in sphingoglycolipids and sphingophospholipids, respectively. Here, those with a sugar bonded are specifically called glycosylceramides, and when the sugar is glucose, they are specifically called glucosylceramides. Ceramides without sugars and phosphoric acid bonded to them are specifically called free ceramides. Furthermore, when the fatty acid portion of ceramide is an ω-hydroxy fatty acid, and another fatty acid is ester-bonded to its hydroxyl terminus, it is specifically called an acylceramide (ω-O-acylceramide). In this specification, unless otherwise specified, acylceramide refers to ω-O-acylceramide. The ceramide compositions of this disclosure preferably contain acylceramides.

[0018] Furthermore, examples of fatty acids that can be esterified to this ω-hydroxy terminus include stearic acid, oleic acid, linoleic acid, and linolenic acid. The ceramide composition of this disclosure (especially the chicken foot ethanol extract ceramide composition) is not particularly limited, but it is preferable that it contains an acylceramide in which the fatty acid is linoleic acid.

[0019] The sphingoid base constituting the free ceramide is preferably one having two or three hydroxyl groups, and more preferably one having three. Furthermore, the sphingoid base is preferably one with 14 to 22 carbon atoms (14, 15, 16, 17, 18, 19, 20, 21, or 22), more preferably one with 16 to 20 carbon atoms, and even more preferably one with 18 or 20 carbon atoms. It is also preferably one with zero or one intercarbon double bond. More specifically preferred sphingoid bases include, for example, sphingosine, dihydrosphingosine, and phytosphingosine.

[0020] The fatty acids constituting free ceramide are preferably those having 16 to 30 carbon atoms (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms), more preferably those having 18 to 28 carbon atoms, even more preferably those having 20 to 26 carbon atoms, and even more preferably those having 22 to 26 carbon atoms. Furthermore, fatty acids having 0 or 1 carbon-carbon double bonds are preferred, and fatty acids having 0 double bonds (i.e., saturated fatty acids) are more preferred. In addition, fatty acids having 0, 1, or 2 hydroxyl groups are preferred. There are no particular limitations, but if hydroxyl groups are present, α-hydroxyl groups are preferred.

[0021] In free ceramides, the (sphingoid base)-(fatty acid) combination of the ceramide skeleton may be any combination of the sphingoid base and fatty acid as described above. Among the preferred combinations, for example, is the combination of (a sphingoid base having 2 or 3 (especially 3) hydroxyl groups)-(a fatty acid having a hydroxyl group (especially an α-hydroxyl group)). The number of hydroxyl groups of the fatty acid is preferably 0, 1, or 2.

[0022] For example, ceramide AS, which is a combination of sphingosine (S) and a fatty acid with one hydroxyl group (A), and ceramide NS, which is a combination of sphingosine (S) and a fatty acid with zero hydroxyl groups (N), are preferred.

[0023] For example, ceramide AP, which is a combination of phytosphingosine (P) and a fatty acid with one hydroxyl group (A), and ceramide NP, which is a combination of phytosphingosine (P) and a fatty acid with zero hydroxyl groups (N), are also preferred examples. Furthermore, ceramide DP, which is a combination of phytosphingosine (P) and a fatty acid with two hydroxyl groups (D), can also be preferred examples. Note that while ceramide AP and ceramide NP are commonly used terms, ceramide DP is a term used in this specification and is not a commonly used term. Specifically, examples of ceramide DP include dihydroxylignoceroylphytosphingosine.

[0024] Among these, the following are preferred as free ceramides: (i) free ceramides in which the ceramide skeleton is a combination of (a sphingoid base having 2 or 3 hydroxyl groups, 18 carbon atoms, and 0 or 1 intercarbon double bond) - (a fatty acid having 16 to 34 carbon atoms, 0 or 1 intercarbon double bond, and 0, 1, or 2 hydroxyl groups); and (ii) free ceramides in which the ceramide skeleton is a combination of (a sphingoid base having 2 or 3 hydroxyl groups, 20 carbon atoms, and no intercarbon double bond) - (a fatty acid having 24 or 25 carbon atoms, 0 intercarbon double bonds, and 0, 1, or 2 hydroxyl groups), with the free ceramide of (i) being more preferred. In particular, free ceramides in which (i-1) the ceramide skeleton is a combination of (a sphingoid base having two hydroxyl groups, 18 carbon atoms, and one carbon-carbon double bond) - (a fatty acid having 16 to 34 carbon atoms, 0 or 1 carbon-carbon double bond, and 0 hydroxyl groups), and (i-2) free ceramides in which the ceramide skeleton is a combination of (a sphingoid base having three hydroxyl groups, 18 carbon atoms, and 0 carbon-carbon double bonds) - (a fatty acid having 20 to 26 carbon atoms, 0 carbon-carbon double bonds, and 0 hydroxyl groups) are preferred. Furthermore, among (i-1), free ceramides in which the ceramide skeleton is a combination of (i-1a) a sphingoid base having two hydroxyl groups, 18 carbon atoms, and one carbon-carbon double bond) and (a fatty acid having 16 to 34 carbon atoms, zero carbon-carbon double bonds, and zero hydroxyl groups) are particularly preferred.

[0025] More specifically, for example, d18:0-16:0, d18:0-18:0, d18:0-20:0, d18:0-22:0, d18:1-16:0, d18:1-18:0, d18:1-20:0, d18:1-22:0, d18:1-24:0, d18:1-26:0, d18:1-26:1, d18:1-28:0, d18:1-30:0, d18:1-32:0, d18:1-32:1, d18:1-34:0, d18:1-34:1, d18:0-16:0h, d18:0-18:0h, d18:0-20:0h, d18:0-22 Examples include :0h, d18:1-16:0h, d18:1-18:0h, d18:1-20:0h, d18:1-22:0h, d18:1-24:0h, d18:1-26:0h, d18:1-28:0h, d18:1-30:0h, d18:1-32:0h, d18:1-34:0h, d18:1-34:1h, t18:0-23:0h, t18:1-23:0h, t18:0-24:0h, t18:1-24:0h, t20:0-24:0h, t18:1-26:0h, t18:0-25:0h, and t18:0-24:0h2. To explain this notation using "t18:0-22:0h" as an example, the first part, "t18:0," provides information about the sphingoid base, indicating a sphingoid base with three hydroxyl groups ("t"), 18 carbon atoms, and zero carbon-carbon double bonds (i.e., no carbon-carbon double bonds). Two hydroxyl groups are represented by "d." The second part, "22:0h," provides information about the fatty acid, indicating a fatty acid with 22 carbon atoms, zero carbon-carbon double bonds, and one hydroxyl group ("h"). "h2" indicates two hydroxyl groups. Note that sometimes "h" is written immediately after the carbon number, as in "22h:0," or "h" is omitted if there are zero hydroxyl groups (conversely, if "h" is not included, it means there are zero hydroxyl groups). Also, sometimes a "c" is placed before the number of carbon atoms, for example, "c22:0".

[0026] Among the ceramide compositions disclosed herein, the chicken foot ethanol extract ceramide composition is preferred because it contains acylceramides. More specifically, the chicken foot ethanol extract ceramide composition is preferred to contain acylceramides of the order d18:1-c28~36:0-18:2 (for example, d18:1-c28:0-18:2, d18:1-c30:0-18:2, d18:1-c32:0-18:2, d18:1-c34:0-18:2, or d18:1-c36:0-18:2), and is particularly preferred to contain four or five of these.

[0027] Ceramides, which constitute the intercellular lipids of the human stratum corneum, have been classified into seven types of free ceramides by Downing et al., depending on the type of long-chain fatty acid linked to the long-chain sphingoid base by an amide bond, and their compositions have also been reported (Robson.KJet al, J.lipid Res. 35, 2060 (1994)). The acylceramide contained in the chicken foot ethanol extract ceramide composition is classified as ceramide EOS among the above seven types. Until now, the only acylceramide prepared by chemical synthesis was ceramide EOP, but ceramide EOP is hardly found in human skin, and rather ceramide EOS is known as the human type. Therefore, the chicken foot ethanol extract ceramide composition is preferable from the standpoint of containing ceramide EOS. Note that ceramide EOS is an acylceramide with sphingosine as the sphingoid base, while ceramide EOP is an acylceramide with phytosphingosine as the sphingoid base.

[0028] The chicken foot ethanol extract ceramide composition may be one in which acylceramide has been concentrated using known methods such as HPLC. In other words, the chicken foot ethanol extract ceramide composition also includes the acylamide concentrate of the chicken foot ethanol extract.

[0029] Furthermore, although not particularly limited, the chicken foot ethanol extract ceramide composition is preferably such that about 0.1 to 2% by mass of the contained ceramide is acylceramide. The upper or lower limit of this range (0.1 to 2% by mass) may be, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9% by mass. For example, the range is more preferably 0.2 to 1.5% by mass, and even more preferably 0.3 to 1.2% by mass. The acylceramide content is a preferred example and may vary, for example, if the acylceramide is concentrated as described above.

[0030] As described above, the chicken foot ethanol extract ceramide composition contains acylceramide (preferably d18:1-c28~36:0-18:2 acylceramide), which is thought to play a very important functional role.

[0031] The acylceramide contained in the chicken foot ethanol extract ceramide composition is of the so-called EOS type, where the sphingoid base is sphingosine and the fatty acid is an esterified ω-fatty acid. Furthermore, the fatty acid esterified with the hydroxyl group of the ω-fatty acid is mainly linoleic acid. In addition, the chain length of the ω-hydroxy fatty acid portion preferably includes 28 to 36 carbon atoms, and the center of its distribution is preferably 30 to 34 carbon atoms.

[0032] Acylceramides, which have very long-chain fatty acid groups, are well known to play a central role in the skin's barrier function, even in small amounts. In particular, a decrease in EOS-type acylceramides, which are found to be present in the intercellular lipids of the human stratum corneum, is known not only to lead to a decline in barrier function and moisturizing function, but also to be an exacerbating factor for skin barrier dysfunction and dry skin in conditions such as atopic dermatitis.

[0033] Furthermore, of the chicken foot ethanol extract ceramide composition, it is preferable that 90% by mass or more of the contained ceramide is ceramide NS, and more preferably 91, 92, 93, 94, 95, 96, 97, or 98% by mass or more is ceramide NS. Furthermore, of the chicken foot ethanol extract ceramide composition, it is preferable that 90% by mass or more of the contained ceramide is the ceramide described in (i-1) above, and more preferably 91, 92, 93, 94, 95, 96, 97, or 98% by mass or more is the ceramide described in (i-1) above. Moreover, of the chicken foot ethanol extract ceramide composition, it is preferable that 90% by mass or more of the contained ceramide is the ceramide described in (i-1a) above, and more preferably 91, 92, 93, or 94% by mass or more is the ceramide described in (i-1a) above.

[0034] Furthermore, although not particularly limited, a preferred chicken foot ethanol extract ceramide composition is one in which d18:1-20:0 is the most abundant, followed by d18:1-22:0, and then d18:1-26:0. For example, while maintaining this order of abundance, a composition is preferred in which d18:1-20:0 is approximately 25-35% by mass (more preferably 27-33% by mass), d18:1-22:0 is approximately 15-30% by mass (more preferably 17-28% by mass), and d18:1-26:0 is approximately 5-20% by mass (more preferably 7-18% by mass or 8-15% by mass).

[0035] Furthermore, d18:1-16:0 is preferably present in 2-8% by mass, and more preferably in 3-7% by mass. d18:1-24:0 is preferably present in 4-11% by mass, and more preferably in 5-10% or 6-9% by mass. d18:1-28:0 is preferably present in 2-8% by mass, and more preferably in 3-7% by mass.

[0036] Furthermore, d18:1-18:0 is preferably present in an amount of 2-6 mass%, and more preferably in an amount of 3-5 mass%. d18:1-26:1 is preferably present in an amount of 0.2-3 mass%, and more preferably in an amount of 0.5-2 mass%. d18:1-30:0 is preferably present in an amount of 1-4 mass%, and more preferably in an amount of 2-3.5 mass%.

[0037] The ceramide composition of this disclosure can also be used in nano-size form (i.e., as a nano-sized ceramide composition). The nano-sized ceramide composition can be obtained, for example, by high-pressure treatment of the ceramide composition. High-pressure treatment can be performed using an ultra-high-pressure wet atomization device (e.g., Sugino Machine Co., Ltd.: Starburst). Examples of high pressure include 100 MPa or more, preferably 100 to 300 MPa, and more preferably 120 to 250 MPa.

[0038] The ceramide compositions of this disclosure can preferably be used as emulsified compositions. In particular, it is preferable that the oil phase is dispersed in the aqueous phase (for example, an oil-in-water (O / W) emulsion). In this case, in addition to bioextracted ceramides (especially chicken foot ethanol-extracted ceramides), the compositions may contain oils and fats, nonionic surfactants, phospholipids, proteins, polysaccharides, pH adjusters, thickeners, fragrances, natural pigments, preservatives, and the like.

[0039] In the case of an O / W emulsion, the average particle size of the ceramide-containing oil droplets (i.e., dispersed particles) is preferably 500 nm or less. From the viewpoint of storage stability over time and bioavailability, the average particle size of the dispersed particles is preferably 300 nm or less, more preferably 200 nm or less, and even more preferably 150 nm or less. There is no particular lower limit to the average particle size of the dispersed particles, but for example, it can be 1 nm or more, and preferably 10, 20, or 50 nm or more.

[0040] In this disclosure, the average particle size of dispersed particles means the volume-average particle size measured using dynamic light scattering.

[0041] Commercially available measuring devices capable of measuring average particle size by dynamic light scattering include the particle size distribution analyzer NanoTrac WAVE II (Verder Scientific), ZetaSizer ULTRA (Malvern), particle size and molecular weight measurement system ELSZ-2000S (Otsuka Electronics), and nanoparticle analyzer SZ-100V2 (Horiba, Ltd.).

[0042] In this specification, the volume-average particle size of dispersed particles can be measured, for example, using a dynamic light scattering method such as NanoTrac WAVE II (Verder Scientific), and specifically, it can be measured as follows: A sample separated from the ceramide dispersion composition of the present invention is diluted with pure water so that the concentration of ceramide contained in the sample is 0.1% by mass, and measured using a quartz cell. The sample refractive index is 1.600, the dispersion medium refractive index is 1.333 (pure water), and the viscosity of the dispersion medium is the viscosity of pure water, and the volume-average particle size (Mv) is determined.

[0043] Furthermore, the average particle size of the dispersed particles containing ceramide can be adjusted not only by the components of the composition, but also by appropriately adjusting the conditions in the manufacturing method described below, such as high-pressure emulsification dispersion conditions (number of passes, pressure, temperature), stirring conditions (shear force, temperature), and the ratio of oil phase to water phase.

[0044] When forming an emulsified composition, the oil phase component and the aqueous phase component may be mixed and pre-dispersed to obtain a crude dispersion beforehand. Such means are not particularly limited, and a general stirring device can be used. Examples of stirring devices include magnetic stirrs, household mixers, paddle mixers, impeller mixers, homomixers, disperser mixers, and ultramixers.

[0045] The time for the preliminary dispersion treatment is not particularly limited and can be set as appropriate depending on the type of stirring device, the composition of the liquid before dispersion treatment, etc.

[0046] The dispersion process can be carried out, for example, by performing a dispersion treatment using ultrasonic dispersion (hereinafter referred to as ultrasonic dispersion) or a dispersion treatment using high-pressure emulsification (hereinafter referred to as high-pressure emulsification) on the crude dispersion obtained in the preliminary dispersion treatment (which may be further mixed with water or other substances as needed).

[0047] In the dispersion process, a crude dispersion containing the bio-extracted ceramide of the present invention (which may be further mixed with water or other substances as needed) is subjected to dispersion to obtain a fine particle ceramide dispersion composition containing dispersed particles that include the bio-extracted ceramide.

[0048] In this dispersion process, the dispersion treatment is preferably carried out by high-pressure emulsification treatment from the viewpoint of miniaturizing the dispersed particles.

[0049] High-pressure emulsification treatment refers to a dispersion treatment in which a shear force of 10 MPa or more is applied to the material to be dispersed. From the viewpoint of miniaturizing dispersed particles, the shear force applied to the material to be dispersed is preferably 50 or 100 MPa or more, and more preferably 150 MPa or more. The upper limit is preferably 300 MPa or less from the viewpoint of temperature rise and pressure resistance.

[0050] The method of high-pressure emulsification is not particularly limited, and general high-pressure emulsification equipment can be used. Examples of high-pressure emulsification equipment include Starburst HJP-25005 (manufactured by Sugino Machinery Co., Ltd.), Microfluidizer (manufactured by Microfluidic Co., Ltd.), Nanomizer (manufactured by Yoshida Machinery Industry Co., Ltd.), Gorin-type homogenizer (manufactured by APV Co., Ltd.), Lannier-type homogenizer (manufactured by Lannier Co., Ltd.), high-pressure homogenizer (manufactured by Nilo Soavi Co., Ltd.), homogenizer (manufactured by Sanwa Machinery Co., Ltd.), high-pressure homogenizer (manufactured by Izumi Food Machinery Co., Ltd.), and ultra-high-pressure homogenizer (manufactured by Ika Co., Ltd.).

[0051] The temperature during the high-pressure emulsification process is preferably set to 30°C to 80°C, and more preferably to 40°C to 70°C.

[0052] While high-pressure emulsification can be performed only once, it is preferable to perform the high-pressure emulsification two or more times, and more preferably two to five times, in order to improve the uniformity of the entire liquid. Furthermore, from the viewpoint of maintaining the particle size of the dispersed particles, it is preferable to cool the emulsified liquid, which is the emulsified and dispersed composition, by passing it through some kind of cooler within 30 seconds, preferably within 3 seconds, immediately after passing it through the chamber.

[0053] In addition to the preliminary dispersion process and the main dispersion process described above, other processes may be included as needed. Examples of other processes include heat sterilization processes.

[0054] Furthermore, the emulsion may be dried to obtain a powdered composition. Powdering offers significant advantages beyond improved long-term storage due to the removal of moisture, including improved portability and reduced transportation costs. Such powdered compositions are also preferably included in the ceramide compositions of this disclosure.

[0055] As drying methods, known drying methods can be used, such as natural drying, heat drying, hot air drying, high-frequency drying, ultrasonic drying, reduced-pressure drying, vacuum drying, freeze-drying, and spray drying. These methods may be used individually, or two or more methods may be used in combination.

[0056] To prevent the aggregation and coalescence of hydrophobic particles during this drying process, a encapsulating agent may be included. Water-soluble polysaccharides and oligosaccharides are preferred as encapsulating agents. The saccharides used as encapsulating agents in this invention are preferably those composed of sugar units as the basic unit. The average degree of polymerization (number of sugar units) of the sugar units is generally preferably 60 or less, and more preferably 2 to 50, from the viewpoint of particle size refinement after condensation. From the viewpoint of stabilizing the coalescence of hydrophobic particles, inulin, raffinose, stachyose, velpascose, and trehalose are preferred as such encapsulating agents.

[0057] Furthermore, in this invention, spray drying is particularly preferred as a drying method from the viewpoint of achieving both production efficiency and quality. Spray drying is a type of convection hot air drying. A liquid emulsion is sprayed into hot air as minute particles of several hundred μm or less, and is recovered as a solid powder as it falls through the tower while being dried. The material is temporarily exposed to hot air, but because the exposure time is very short and the temperature does not rise much due to the latent heat of evaporation of water, thermal deformation of the material is unlikely to occur, similar to freeze drying, and changes due to condensate are also small. In the case of a material that is very sensitive to heat, it is also possible to supply cold air instead of hot air. In that case, although the drying capacity is reduced, it is preferable in that a milder drying can be achieved.

[0058] Examples of commercially available spray dryers include, but are not limited to, the Spray Dryer SD-1000 (Tokyo Rikakikai Co., Ltd.), Spray Dryer L-8i (Okawara Kakoki Co., Ltd.), Closed Spray Dryer CL-12 (Okawara Kakoki Co., Ltd.), Spray Dryer ADL310 (Yamato Kagaku Co., Ltd.), Mini Spray Dryer B-290 (Büch GmbH), PJ-MiniMax (Powdering Japan Co., Ltd.), and PHARMASD (Niro Co., Ltd.).

[0059] Furthermore, devices that can perform drying and granulation simultaneously, such as the MP-01 fluidized bed granulator (Powrec Co., Ltd.) and the FSD fluidized bed spray dryer (Niro Co., Ltd.), are also preferable.

[0060] As described above, the ceramide composition of this disclosure can be used as a topical composition, an oral composition, an intestinal composition, etc. It can also be preferably used in the pharmaceutical and food fields. As detailed below, the composition may consist only of ceramide, or it may be a composition containing ceramide and other components (various bases, carriers, additives, etc.). Ceramide-containing biological tissue extract itself is also included in the composition. In other words, ceramide-containing biological tissue extract (and, if necessary, further compounded with other components) can also be used as the ceramide composition of this disclosure.

[0061] When the ceramide composition of this disclosure is used in the pharmaceutical field (including pharmaceuticals and quasi-drugs), the composition (hereinafter sometimes referred to as "pharmaceutical composition relating to this disclosure") may consist only of each bioextracted ceramide, or it may be a pharmaceutical composition containing other components. For example, in the pharmaceutical composition according to the present invention, pharmaceutically acceptable bases, carriers, and additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavoring agents, odoring agents, surfactants (especially emulsifiers), humectants, preservatives, pH adjusters, viscosity modifiers, etc.) may be added to the ceramide, which is the active ingredient, as needed. Such bases, carriers, and additives are specifically described in, for example, the Pharmaceutical Additives Dictionary 2016 (Yakuji Nippo Co., Ltd.), and those described therein can be used. Furthermore, there are no particular restrictions on the formulation form. The active ingredient and other ingredients can be mixed by conventional methods, and for example, oral compositions can be prepared as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewables, soft tablets, etc. For example, tablets can be manufactured by tableting. Either direct tableting, in which the mixed raw materials are compressed as is, or granular tableting, in which the mixed raw materials are granulated before tableting, can be used. Also, for example, in the case of capsules, either soft capsules or hard capsules may be used. Also, for example, external compositions can be used as creams, emulsions, gels, etc. Also, for example, compositions for intestinal administration can be used as foams, for example.

[0062] The amount of ceramide in the pharmaceutical composition according to the present invention is not particularly limited as long as an anti-fatigue effect is exerted, and can be set appropriately according to the target person. Preferably, it is 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be around 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by mass.

[0063] The subjects to whom the pharmaceutical compositions described herein can be administered include not only humans but also non-human mammals. Examples of mammals that exhibit fatigue include, and mammals kept as pets and livestock are particularly preferred. Specifically, examples include dogs, cats, monkeys, cattle, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, and the like. In the case of mammals, as in the case of humans, the pharmaceutical compositions according to the present invention can also be used preventively.

[0064] The timing of administration of the pharmaceutical composition relating to this disclosure is not particularly limited, and the timing of administration can be appropriately selected considering, for example, the form of formulation, the age of the recipient, the severity of the symptoms of the recipient, etc.

[0065] The dosage of the pharmaceutical composition relating to this disclosure can be appropriately selected according to the age of the recipient, the severity of the symptoms, and other conditions. In particular, it can be appropriately set based on the amount of ceramide contained, within a range that does not impair the effects of the present invention. It can be administered once a day or divided into multiple doses (preferably 2 to 3 times). In the case of mammals, the dosage can also be appropriately set with reference to the case of humans.

[0066] When the ceramide composition of this disclosure is used as a food composition (e.g., food or beverage or food additive), the composition (hereinafter sometimes referred to as "food composition of this disclosure") is a mixture of ceramide and food hygiene-acceptable bases, carriers, additives, and other components and materials that can be used as food or beverages. Examples of food compositions containing ceramide include processed foods, beverages, health foods (nutrient function foods, foods for specified health uses, etc.), supplements, and foods for the sick (hospital food, sick person's food, or nursing care food, etc.) for improving or preventing fatigue. Although not particularly limited, if the ceramide incorporated into the food composition is a bio-extracted ceramide extracted from biological (especially animal or plant) tissue, it is preferable that the food composition contains such ceramide, for example, processed foods, health foods (nutrient function foods, foods for specified health uses, etc.), supplements, or foods for the sick. Furthermore, ceramide may be incorporated into various foods and beverages, such as drinks (juices, etc.), confectionery, bread, soups (including powdered soups, etc.), and processed foods, for example, by being in powder form.

[0067] When preparing the food composition according to the present invention as a health food (nutrient functional food, food for specified health uses, etc.) or supplement, it is preferable to prepare it in the form of granules, capsules, tablets (including chewable tablets, etc.), beverages (drinks), etc., to facilitate continuous intake. Among these, capsules, tablets, and granules are preferred in terms of ease of intake, but the invention is not limited to these. The food composition according to the present invention in the form of granules, capsules, tablets, etc., can be appropriately prepared according to conventional methods using pharmaceutically and / or food hygiene acceptable carriers, etc. Furthermore, even when preparing it in other forms, conventional methods may be followed.

[0068] The amount of ceramide in the food composition according to this disclosure is not particularly limited as long as the effect can be achieved. Preferably, it is 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass. The lower limit may be around 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by mass.

[0069] The amount ingested, the target of intake, etc., of the food composition relating to this disclosure are not particularly limited, but are preferably the same as those of the pharmaceutical composition according to the present invention described above.

[0070] Hospital meals are meals served to patients admitted to a hospital, sick person meals are meals for sick people, and nursing care meals are meals for people receiving nursing care.

[0071] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Furthermore, this disclosure encompasses all any combination of the constituent elements described herein.

[0072] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0073] The present invention will be described in more detail below, but it is not limited to the following examples. Preparation of ceramide compositions <Soy sauce lees extract ceramide composition> Based on the method described in Japanese Patent Publication No. 2012-126910, soy sauce lees were extracted with ethanol, and high-purity free ceramide was purified by solvent fractionation to obtain a ceramide composition.

[0074] More specifically, the preparation was carried out as follows: Ethanol extraction was performed on the dried lees, a by-product of soy sauce produced by conventional methods. The resulting extract was separated into solid and liquid components using ethanol and water to obtain a solid portion. This solid portion was then washed with acetone, ethanol, and water, dried, and pulverized, and then washed again with water, acetone, and hexane. The resulting solid portion was then extracted with ethanol again to obtain a ceramide composition.

[0075] <Aspergillus oryzae-extracted ceramide composition> Mold cells were collected from 300 liters of Aspergillus oryzae culture solution and dried to obtain a solid. A 2.5-fold ethanol / water mixture was added to the solid and mixed, and the mixture was stirred and extracted overnight at 40°C. After collecting the extract, a 1.2-fold ethanol solution was added to the remaining solid and mixed, and the mixture was stirred and extracted overnight at 40°C. The resulting extracts were combined and distilled under reduced pressure at 45°C to obtain a concentrated extract. After washing the concentrated extract with acetone (and further washing with ethanol, acetone, etc., as needed), the resulting insoluble material was used as the Aspergillus oryzae extract ceramide composition.

[0076] <Chicken foot extract ceramide composition> Dried chicken was mixed with 2.5 times the volume of 95% ethanol and extracted overnight at 40°C with stirring. After collecting the extract, 1.2 times the volume of ethanol was added to the solid portion and mixed, and extracted overnight at 40°C with stirring. The resulting extracts were combined and distilled under reduced pressure at 45°C to obtain a concentrated extract. After washing the concentrated extract with acetone (and further washing with ethanol, acetone, etc., as needed), the resulting waste was used as the chicken foot extract ceramide composition. More specifically, this chicken foot extract ceramide composition can be called a chicken foot ethanol extract ceramide composition.

[0077] Compositional analysis of ceramide compositions by LC-MS Each ceramide composition was analyzed using LC-MS (high-performance liquid chromatography-mass spectrometry) to determine the various free ceramide species contained in each composition.

[0078] Figure 15 and Table 1 show the content percentages (by mass) of various free ceramide species in the soy sauce lees extract ceramide composition. Three types of free ceramide species, t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h, accounted for more than 50% by mass of the ceramide composition. In addition, it was found that other free ceramides were also present. [Table 1]

[0079] Furthermore, the free ceramide molecular species of the Aspergillus oryzae-extracted ceramide composition are shown in Table 2. [Table 2]

[0080] Furthermore, four batches of chicken foot extract ceramide composition were prepared and analyzed for the various free ceramide species contained. The results are shown in Tables 3a and 3b. Table 3a shows the content (mg / g) of each free ceramide, and Table 3b shows the concentration (mass%) of each free ceramide. [Table 3a]

[0081] [Table 3b]

[0082] In the table, (d18:1-c28:0-18:2) (d18:1-c30:0-18:2)(d18:1-c32:0-18:2)(d18:1-c34:0-18:2)(d18:1-c36:0-18:2) represent acylceramides.

[0083] Nano-sized ceramides Glycols (other polyols, oils, etc. may be added as needed), an emulsifier, and a ceramide composition were mixed and heated to 100-150°C (temperature near the melting point of ceramide) to make a homogenized mixture. A lecithin aqueous dispersion, a polysaccharide aqueous solution, a water-soluble polymer aqueous solution, etc., were further added and stirred and mixed. The resulting mixture was processed at a high pressure of 100 MPa or more using an ultra-high pressure wet atomization device (Sugino Machine Co., Ltd.: Starburst) to nano-size the ceramide. Lecithin was used in a mass ratio of approximately 2 to 5 times that of ceramide.

[0084] Furthermore, the particle size of ceramide-containing particles in the obtained nano-sized ceramide composition was measured using the dynamic light scattering method NanoTrack WAVE II (Verder Scientific). Specifically, the nano-sized ceramide composition (emulsion) was diluted with pure water to a ceramide concentration of 0.1 mass%, and this was used as the measurement sample. The sample refractive index was 1.600, the dispersion medium refractive index was 1.333 (pure water), and the viscosity of pure water was used as the viscosity of the dispersion medium to determine the volume-average particle size (Mv). The ceramide particle size was also measured in the same manner for the ceramide composition before nano-processing. The volume-average particle size of ceramide in the ceramide composition before nano-processing was 4857 nm, and the volume-average particle size of ceramide in the nano-sized ceramide composition was 124 nm.

[0085] In the following, the soy sauce lees-extracted ceramide composition was nano-sized and used as the nano-sized ceramide composition.

[0086] Each ceramide composition (soy sauce lees extract ceramide composition, Aspergillus oryzae extract ceramide composition, chicken foot extract ceramide composition, nano-sized ceramide composition), as well as glucosylceramide and synthetic ceramide, were used as ceramide samples. In the following studies, the ceramide concentration refers to the concentration of each ceramide sample. In the following descriptions, "○○ extract ceramide composition" may be abbreviated as "○○ ceramide." For example, soy sauce lees extract ceramide composition may be written as soy sauce lees ceramide. Also, chicken foot extract ceramide composition may be written as Avian. For glucosidoceramide, Maruzen Pharmaceutical Co., Ltd. (Pine Cera Powder) was used; for synthetic ceramide, Takasago International Corporation (Ceramide NG "CERAMIDE TIC-001") was used; and for NAC, Fujifilm Wako (N-acetyl-L-cysteine) was used.

[0087] [Method of Consideration] <Evaluation of ceramide's barrier function enhancement in HaCaT cells> HaCaT cell culture The human epidermal keratinocyte line HaCaT cells were used as a model cell for human skin. The cells were subcultured in DMEM medium (Dulbecco's Modified Eagle Medium; Nissui, Tokyo, Japan) supplemented with inactivated 10% fetal bovine serum (FBS; Life Technologies, CA, USA) in cell culture dishes (Greiner bio-one, Tokyo, Japan) at 37°C in the presence of 5% CO2. DMEM medium was prepared by dissolving 4.75 g of Dulbecco's Modified Eagle Medium (DMEM) medium "Nissui" (2) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) in 470 mL of Milli-Q water, and adding 10 mL of 0.2 M L-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), 0.5 mL of 0.05 mg / mL streptomycin (Meiji Seika Pharma Co., Ltd.), and 6 mL of 10% NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0088] Mitochondrial activity measurement using IN Cell Analyzer 2200 HaCaT cells 6.0 × 10 4 Cells were seeded at a density of cells / mL in a 96-well plate and pre-cultured for 24 hours. Subsequently, ceramide was added to a final concentration of 5 μM. After 24 hours, 100 μL / well of MitoTracker Red (Invitrogen, USA), diluted to 2.5 μM in 10% FBS / DMEM, was added to the plate after removing the medium, and incubated at 37°C under 5% CO2 conditions for 30 minutes. After 30 minutes, the medium containing MitoTracker Red was removed, and 100 μL / well of MitoTracker Green (Invitrogen), diluted to 200 nM in 10% FBS / DMEM, was added, and incubated at 37°C under 5% CO2 conditions for 30 minutes. Subsequently, 100 μL / well of a 1 mg / mL Cellstain Hoechst 33342 solution (DOJINDO, Kumamoto, Japan), diluted 500-fold in DMEM, was added, and incubated at 37°C under 5% CO2 conditions for 30 minutes. The diluted Hoechst 33345 solution was then removed from the 96-well plate, 150 mL of PBS was added, and fluorescence detection was performed using an IN Cell Analyzer 2200 (GE Healthcare). The activity, number, and area of ​​mitochondria within the cells were investigated by measuring the fluorescence levels of Mito Tracker Red, Mito Tracker Green, and Hoechst 33342. Images were analyzed using IN Cell Investigator High-content image analysis software (GE Healthcare).

[0089] ROS measurement using IN Cell Analyzer 2200 Subconfluent HaCaT cells seeded in a 10 mL dish were subjected to UVB 10 mJ / cm² radiation in a UV crosslinker (CL-1000 Ultraviolet Crosslinker, UVP, Upland, CA, USA) with the dish lid removed. 2 Irradiation was performed using UVB, and immediately afterward, 10 mL of 10% FBS-containing DMEM medium was added and incubated at 37°C in the presence of 5% CO2. Untreated cells were not irradiated with UVB, and only the medium was changed. After 24 hours, the culture media of the non-UVB and UVB irradiated cells were discarded, washed with 1×PBS, and the cells were detached with trypsin and placed in 3×10⁶ wells of a 96-well plate. 3 The cells were seeded to form cells. After 4 hours, they were treated with ceramide and incubated for 24 hours. Subsequently, intracellular ROS production in HaCaT cells was measured using BES-H2O2-AC (Wako Pure Chemical Industries) according to the manufacturer's protocol. The culture medium in the 96-well plate was removed, and the cells were washed twice with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer (pH 7.4). Cellstain®-Hoechst 33342 solution (Dojindo) was mixed with 1×PBS in a ratio of 1:500 and applied to the cells, which were then left to stand in the dark at room temperature for 30 minutes. Subsequently, 5 μM BES-H2O2-AC in HEPES (pH 7.4) was mixed with Hoechst 33342 (Dojindo) in a 1:1000 ratio and incubated with cells at 37°C for 60 minutes. The protocol "HaCaT_eGFP20190627" was used, and images were acquired using an IN Cell Analyzer 2200 (GE Healthcare). The images were analyzed using an IN Cell Analyzer 2200 Workstation (GE Healthcare) to examine ROS expression levels.

[0090] The UV damage recovery effect of ceramides in UVB-induced senescent cells. Subconfluent cells are exposed to UVB at a rate of 10 mJ / cm². 2Using irradiated cells, the same experiment as described in "Measurement of Mitochondrial Activity Using IN Cell Analyzer 2200" was performed.

[0091] Evaluation of human ceramide synthesis in HaCaT cells HaCaT cells cultured in a 10 mL dish in a subconfluent state were treated with ceramide sample to a final concentration of 5 μM. After 48 hours, the medium was removed, washed with 5 mL of 1×PBS, and 1.5 mL of trypsin was added to permeate the entire dish before removal. The cells were incubated at 37°C under 5% CO2 conditions for 8 minutes, detached using DMEM, collected in a 15 mL tube, and centrifuged at 12000 rpm for 3 minutes to collect a cell pellet. The fatty acid composition of ceramide contained in the HaCaT cells was analyzed using LC-MS.

[0092] <Evaluation of the effects of ceramide on HaCaT cells via Caco-2 cells: Examination of barrier function in the intestinal tract> Caco-2 cell culture As a human intestinal epithelial cell model, we used the human colon cancer-derived cell line CaCo-2 cells. CaCo-2 cells were subcultured in a cell culture dish (Greiner bio-one, Tokyo, Japan) at 37°C in the presence of 5% CO2 using DMEM medium containing 10% inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating in a 56°C incubator for 35 minutes). DMEM medium was prepared by dissolving 4.75 g of Dulbecco's Modified Eagle Medium (DMEM) medium "Nissui" (2) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) in 470 mL of Milli-Q water, and adding 10 mL of 0.2 M L-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), 0.5 mL of 0.05 mg / mL streptomycin (Meiji Seika Pharma Co., Ltd.), and 6 mL of 10% NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0093] Mitochondrial activity measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells were seeded at 1.0×10 2 cells / mL in a 24-well plate (Corning, NY, USA). After 24 hours, ceramide at a final concentration of 5 μM was added and cultured for 24 hours. 6.0×10 4 cells / mL were seeded in a 96-well plate, 50 μL of the culture medium of HaCaT cells incubated for 24 hours was removed, and 50 μL of the culture medium of Caco-2 cells was added thereto. 48 hours later, the number and area of mitochondria and the mitochondrial membrane potential activity of HaCaT cells were measured by the same method as described in the above "Measurement of Mitochondrial Activity Using IN Cell Analyzer 2200".

[0094] ROS measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells were seeded at 1.0×10 2 cells / mL in a 24-well plate (Corning, NY, USA). After 24 hours, ceramide at a final concentration of 5 μM was added and cultured for 24 hours. 6.0×10 4 cells / mL were seeded in a 96-well plate, 50 μL of the culture medium of HaCaT cells incubated for 24 hours was removed, and 50 μL of the culture medium of Caco-2 cells was added thereto. 48 hours later, the ROS of HaCaT cells was measured by the same method as described in the above "Measurement of ROS Using IN Cell Analyzer 2200".

[0095] The UV damage recovery effect of ceramides in UVB-induced senescent cells (Caco-2 cells → HaCaT cells) Caco-2 cells were seeded at 1.0×10 2 cells / mL in a 24-well plate (Corning, NY, USA). After 24 hours, ceramide at a final concentration of 5 μM was added and cultured for 24 hours. Sub-confluent HaCaT cells were irradiated with UVB at 10 mJ / cm 2 and 6.0×10 4Cells were seeded in a 96-well plate at a concentration of cells / mL. After 24 hours, 50 μL of the HaCaT cell culture was removed, and 50 μL of the Caco-2 cell culture was added. After 48 hours, the number and area of ​​mitochondria and mitochondrial membrane potential activity of HaCaT cells were measured using the same method as described in "The UV damage recovery effect of ceramide in UVB-induced senescent cells".

[0096] Transepithelial Electrical Resistance (TER) measurement A Cell Culture Insert (FALCON) was placed in a 24-well plate. Caco-2 cells were placed in a 2.0 × 10⁶ well. 5 Cells were seeded in a single well, and carnosine was added to a concentration of 10 mM after 48 hours. Additions were made every 3 days, and the cells were cultured for 2 weeks. A Millicell ERS-2 (Millipore Japan, Tokyo) was used to measure transepithelial electrical resistance. Before the experiment, electrodes were set in the Millicell ERS-2, sterilized by immersing them in a 70% ethanol solution for 10 minutes, then dried and immersed in DMEM culture medium. Next, electrodes were inserted into the inside and outside of a Cell Culture Insert, and measurements were taken. The measured values ​​were calculated using the following formula as a reference. TER (Ω·cm 2 ) = (measured value - blank) × (insert area)

[0097] [Results of the review] Mitochondrial activity measurement using IN Cell Analyzer 2200 The number of mitochondria, area (Mitotracker Green), and mitochondrial membrane potential activity (Mitotracker Red) per cell were measured using an IN Cell Analyzer 2200 after treating HaCaT cells with ceramides for 48 hours. Glycosylceramide, synthetic ceramide, soy sauce lees ceramide, Aspergillus oryzae ceramide, Avian ceramide, and non-nano-sized ceramide significantly increased the number of mitochondria in HaCaT cells. Glycosylceramide, soy sauce lees ceramide, Aspergillus oryzae ceramide, and Avian ceramide significantly increased the mitochondrial area in HaCaT cells. (Figure 1)

[0098] ROS measurement using IN Cell Analyzer 2200 HaCaT cells: 10 mJ / cm² 2 After UVB irradiation, ROS levels were measured using an IN Cell Analyzer 2200 after 24-hour treatment with ceramide. The results showed that only Aspergillus oryzae ceramide significantly reduced ROS. (Figure 2)

[0099] Mitochondrial activity recovery effect as measured by IN Cell Analyzer 2200 HaCaT cells: 10 mJ / cm² 2 After UVB irradiation, the number and area of ​​mitochondria per cell (Mitotracker Green) and mitochondrial membrane potential activity (Mitotracker Red) were measured using an IN Cell Analyzer 2200 after 48 hours of treatment with ceramide. (Figure 3)

[0100] Evaluation of human-type ceramide synthesis from natural human-type ceramides using LC-MS HaCaT cells in a subconfluent state were treated with ceramide for 48 hours, and the fatty acid composition of the HaCaT cells was measured using LC-MS. As a result, in HaCaT cells treated with soy sauce lees ceramide, Aspergillus oryzae ceramide, and Avian ceramide, the amount of human-type ceramides with 24 carbon atoms in long-chain fatty acids was significantly increased. (Figure 4)

[0101] Mitochondrial activity measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells were treated with ceramide for 24 hours, and the supernatant was added to HaCaT cells and treated for 48 hours. The number and area of ​​mitochondria per cell (Mitotracker Green) and mitochondrial membrane potential activity (Mitotracker Red) were measured using an IN Cell Analyzer 2200. (Figure 5)

[0102] ROS measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells were treated with ceramide for 24 hours, and the supernatant was taken at 10 mJ / cm³. 2The ROS levels were measured using an IN Cell Analyzer 2200 after adding the specified substances to HaCaT cells irradiated with UVB and treating them for 24 hours. The results showed that both Aspergillus oryzae ceramide and nano-sized ceramide significantly reduced ROS levels. (Figure 6)

[0103] Mitochondrial activity restoration effect (Caco-2 cells → HaCaT cells) as measured by IN Cell Analyzer 2200. Caco-2 cells were treated with ceramide for 24 hours, and the supernatant was taken at 10 mJ / cm³. 2 The number and area of ​​mitochondria per cell (Mitotracker Green) and mitochondrial membrane potential activity (Mitotracker Red) were measured using an IN Cell Analyzer 2200 after adding the solution to HaCaT cells irradiated with UVB and treating them for 24 hours. (Figure 7)

[0104] Evaluation of barrier function enhancement by natural human-type ceramides in CaCo-2 cells Measurement of endogenous Claudin3 expression levels (Caco-2 cells) Endogenous Claudin3 expression levels were measured by quantitative RT-PCR after treating Caco-2 cells with ceramide for 24 hours. The results showed a significant enhancement of Claudin3 expression in Caco-2 cells treated with glycosylceramide and avianceramide. (Figure 8)

Claims

1. A chicken foot ethanol-extracted ceramide composition for strengthening the epithelial barrier.

2. A chicken foot ethanol-extracted ceramide composition for strengthening the skin barrier or the intestinal barrier.

3. A skin barrier strengthening composition according to claim 2, for caring for skin damage caused by ultraviolet rays.

4. The intestinal barrier strengthening composition according to claim 2, for strengthening tight junctions of intestinal epithelial cells.

5. A composition according to any one of claims 1 to 4, comprising ω-O-acylceramide.

6. The composition according to claim 5, wherein the contained ω-O-acylceramide is of the EOS type and the terminal acyl group is a linoleic acid group.

7. The composition according to claim 5, wherein 0.1 to 2% by mass of the ceramide contained is ω-O-acylceramide.

8. The composition according to any one of claims 1 to 4, wherein 90% by mass or more of the ceramide contained is free ceramide whose ceramide skeleton is a combination of (a sphingoid base having two hydroxyl groups, 18 carbon atoms, and one carbon-carbon double bond) - (a fatty acid having 16 to 34 carbon atoms, 0 or 1 carbon-carbon double bond, and 0 hydroxyl groups).

9. The composition according to any one of claims 1 to 4, wherein 90% by mass or more of the ceramides contained are ceramide NS.

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