Ceramide composition

Abstract

To find new applications for ceramide compositions. [Solution] A composition for activating sirtuin genes, comprising a ceramide composition, The ceramide composition is at least one bio-extracted ceramide composition selected from the group consisting of soy sauce lees extract ceramide composition, koji mold extract ceramide composition, and chicken foot extract ceramide composition. composition.

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 Initiative] [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 by adding specific structures, such as nano-sizing, to these ceramide compositions, they may be able to perform these specific functions even more efficiently. Based on these findings, further improvements have been made.

[0007] This disclosure includes, for example, the following subjects: Section 1. A composition for activating sirtuin genes, comprising a ceramide composition, The ceramide composition is at least one bio-extracted ceramide composition selected from the group consisting of soy sauce lees extract ceramide composition, koji mold extract ceramide composition, and chicken foot extract ceramide composition. composition. Section 2. A composition comprising a ceramide composition for use as a skin barrier, an intestinal barrier, or for anti-skin aging, The ceramide composition is at least one bio-extracted ceramide composition selected from the group consisting of soy sauce lees extract ceramide composition, koji mold extract ceramide composition, and chicken foot extract ceramide composition. composition. Section 3. The composition according to item 2, which is for use as a skin barrier or for anti-skin aging. Section 4. A composition for caring for skin damage caused by ultraviolet rays, as described in item 3. Section 5. The composition described in item 2, for use as an intestinal barrier. Section 6. The composition according to item 5, for strengthening tight junctions in the intestinal tract. Section 7. The composition according to any one of claims 1 to 6, comprising at least t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h. Section 8. The total amount of t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h is 30% by mass or more of the total amount of free ceramide contained. The composition according to item 7. Item 9. The composition according to any one of items 1 to 8, wherein the ceramide composition is an O / W type emulsion composition and the volume average particle diameter of the dispersed oil phase particles is 10 to 500 nm.

Advantages of the Invention

[0008] There is provided a composition (a specific ceramide-containing composition) that can efficiently activate the sirtuin gene. Further, the composition is also useful as a skin barrier, an intestinal barrier, or an anti-skin aging agent.

Brief Description of the Drawings

[0009] [Figure 1] The results of measuring the mRNA expression level of SIRT1 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 the mRNA expression level of SIRT3 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 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 and culturing for 48 hours are shown. (n = 3; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t-test) [Figure 4] 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 5]The following shows the results of measuring mitochondrial number, area, and membrane potential activity using an IN Cell Analyzer 2200 after adding ceramide to HaCaT cells irradiated with 10 mJ / cm2 UVB and culturing for 24 hours. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 6] The results of analyzing the fatty acid composition of ceramide in HaCaT cells using LC-MS after adding ceramide and culturing for 48 hours are shown. [Figure 7] The following shows the results of measuring SIRT1 mRNA expression levels after adding the supernatant of Caco-2 cells treated with ceramide for 24 hours and culturing them for 48 hours. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 8] The following shows the results of measuring SIRT3 mRNA expression levels after adding the supernatant of Caco-2 cells treated with ceramide for 24 hours and culturing them for 48 hours. (n=3; *p<0.05, #p<0.15 Student's t-test) [Figure 9] The following results show the mitochondrial number, area, and membrane potential activity measured 48 hours after adding the supernatant of Caco-2 cells treated with ceramide for 24 hours to HaCaT cells irradiated with 10 mJ / cm2 UVB. (n=3; ***p <0.001, **p<0.01, *p<0.05,#=0.08 Student's t-test) [Figure 10] The following shows the results of measuring ROS (reactive oxygen species) after 48 hours using an IN Cell Analyzer 2200, following the addition of supernatant from Caco-2 cells treated with ceramide for 24 hours to HaCaT cells irradiated with 10 mJ / cm2 UVB. (n=3; ***p <0.001, **p<0.01, *p<0.05, #p<0.15, Student's t-test) [Figure 11]The following results show the measurements of mitochondrial number, area, and membrane potential activity after 48 hours, using an IN Cell Analyzer 2200, following the addition of supernatant from Caco-2 cells treated with ceramide for 24 hours to HaCaT cells irradiated with 10 mJ / cm2 UVB. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 12] The following shows the results of measuring SIRT1 mRNA expression levels after adding ceramide to Caco-2 cells and culturing them for 24 hours. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 13a] The following shows the results of measuring Claudin-3 mRNA expression levels after adding ceramide to Caco-2 cells and culturing them for 24 hours. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 13b] The following shows the results of measuring ZO-1 mRNA expression levels after adding ceramide to Caco-2 cells and culturing them for 24 hours. (n=3; ***p <0.001, **p<0.01, *p<0.05, Student's t-test) [Figure 14a] The results of measuring TER values ​​every 24 hours after adding ceramide to Caco-2 cells are shown. [Figure 14b] The results of measuring TER values ​​every 24 hours after adding ceramide to Caco-2 cells are shown. [Figure 15] This shows the percentage (by mass) of various free ceramide species contained in the soy sauce lees extract ceramide composition. [Modes for carrying out the invention]

[0010] The embodiments included in this disclosure will be described in further detail below. This disclosure preferably includes, but is not limited to, certain bio-derived ceramide compositions and their uses, and encompasses everything disclosed herein and recognizable to those skilled in the art.

[0011] 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 ceramides. These bio-derived ceramides can be used individually or in combination of two or more. Such bio-derived ceramides may be referred to as the ceramide compositions of this disclosure. The term "ceramide composition" herein means a composition that contains ceramides.

[0012] The ceramide composition disclosed herein can be preferably used to activate sirtuin genes. Sirtuin genes are also called anti-aging genes, and it is believed that their activation produces anti-aging effects. The ceramide composition disclosed herein can be preferably used to activate sirtuin genes in mammals (especially humans). Furthermore, among sirtuin genes, it can be preferably used to activate the SIRT1 gene (NCBI Gene ID: 23411) and the SIRT3 gene (NCBI Gene ID: 23410).

[0013] Furthermore, when the ceramide composition of this disclosure is used for sirtuin gene activation, it can be used, for example, as a topical composition or an oral composition (for example, an oral pharmaceutical composition or a food composition).

[0014] While not particularly limited, when used as a topical composition, for SIRT1 gene activation, soy sauce lees extract ceramide composition and / or chicken foot extract ceramide are particularly preferred among naturally derived ceramide compositions. Furthermore, for SIRT3 gene activation, a composition containing nano-sized ceramide (nano-sized ceramide composition) is preferred. Furthermore, for SIRT3 gene activation, chicken foot extract ceramide composition is particularly preferred among naturally derived ceramide compositions.

[0015] Furthermore, although not particularly limited, when used as an oral composition, it is preferable that the composition contains nano-sized ceramides (nano-sized ceramide composition) for the purpose of activating the SIRT1 gene and / or the SIRT3 gene. In addition, for the purpose of activating the SIRT3 gene, among bio-derived ceramide compositions, a ceramide composition extracted from Aspergillus oryzae is particularly preferred.

[0016] Furthermore, the ceramide compositions of this disclosure can be preferably used for skin or intestinal barriers. They can also be preferably used for anti-aging purposes.

[0017] When the ceramide compositions of this disclosure are used for skin barrier or anti-aging purposes, they are particularly preferable for preventing the effects of ultraviolet (UV) 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 enhance the expression of sirtuin genes (particularly SIRT1 and SIRT3 genes), which are known to be involved in cell repair and reactive oxygen species (ROS) scavenging, in skin cells damaged or affected by UV radiation. Furthermore, applying the ceramide compositions of this disclosure to the skin may restore mitochondria (particularly in number, area, or membrane potential activity) in skin cells damaged or affected by UV radiation. It can be used preventively for UV-damaged skin, or therapeutically for UV-damaged skin.

[0018] Furthermore, when the ceramide composition of this disclosure is used for skin (for skin barrier or anti-aging purposes), it can be used, for example, as a topical composition or an oral composition (for example, an oral pharmaceutical composition or a food composition). In addition, when the ceramide composition of this disclosure is used for skin, it can enhance the SIRT3 gene, and therefore a whitening effect can also be expected.

[0019] When the ceramide composition of this disclosure is used for the intestinal barrier, it is particularly preferable to use it to strengthen tight junctions in the intestinal tract. Although we do not wish to be bound by theory, this is because applying the ceramide composition of this disclosure to the intestinal tract may enhance the expression of tight junction genes (particularly the ZO-1 gene) 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.

[0020] Furthermore, the density and coupling properties of tight junctions can also be measured using trans-epithelial electrical resistance (TER) as an indicator, as disclosed in this disclosure. The ceramide composition can preferably improve the TER value.

[0021] Furthermore, when the ceramide composition of this disclosure is used in the intestinal tract (for use 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 a composition for intestinal administration.

[0022] Furthermore, when the ceramide composition of this disclosure is used to strengthen intestinal tight junctions (particularly to enhance ZO-1 gene expression), it is preferable that the composition contains nano-sized ceramides (nano-sized ceramide composition). In this case, among the bio-derived ceramide compositions, soy sauce lees-extracted ceramide compositions and / or Aspergillus oryzae-extracted ceramide compositions are particularly preferable.

[0023] 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 phosphates 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. When ceramide does not have sugar or phosphate bonded to it, it is specifically called free ceramide. To refer to it. Free ceramides are preferred as the ceramides contained in the ceramide composition of this disclosure.

[0024] 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.

[0025] 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. Additionally, fatty acids having 0, 1, or 2 hydroxyl groups are preferred, and fatty acids having 1 or 2 hydroxyl groups are more preferred. There are no particular limitations, but if hydroxyl groups are present, α-hydroxyl groups are preferred.

[0026] 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 (particularly 3) hydroxyl groups)-(a fatty acid having a hydroxyl group (particularly an α-hydroxyl group)). The number of hydroxyl groups of the fatty acid is preferably 0, 1, or 2, and more preferably 1 or 2.

[0027] 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 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 herein and is not a commonly used term. Specifically, examples of ceramide DP include dihydroxylignoceroylphytosphingosine.

[0028] 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 3 hydroxyl groups, 18 carbon atoms, and 0 or 1 intercarbon double bond) - (a fatty acid having 22 to 26 carbon atoms, 0 intercarbon double bonds, and 0, 1, or 2 hydroxyl groups); and (ii) free ceramides in which the ceramide skeleton is a combination of (a sphingoid base having 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). In particular, more specifically, t18:0-22:0h, t18:1-22:0h, 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 are preferred. To explain this notation using "t18:0-22:0h" as an example, the first part "t18:0" is information about the sphingoid base, which has three hydroxyl groups ("t"), 18 carbon atoms, and zero carbon-carbon double bonds (i.e., no carbon-carbon double bonds). Furthermore, the latter part, "22:0h," provides information about the fatty acid, indicating that it is a fatty acid with 22 carbon atoms, 0 carbon-carbon double bonds, and one hydroxyl group ("h"). Note that "h2" indicates the presence of two hydroxyl groups. In some cases, "h" is written immediately after the carbon number, as in "22h:0."

[0029] Furthermore, the ceramide composition of this disclosure preferably includes t18:0-22:0h, t18:1-22:0h, 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, and is particularly preferably composed of t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h. It is also preferable that at least t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h are included as free ceramides. In particular, it is more preferable that the total amount of t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h relative to the total amount of free ceramide contained in the composition is 30% by mass or more, 40% by mass or more, or 50% by mass or more. It may also be 60% by mass or more, or 70% by mass or more.

[0030] 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.

[0031] The ceramide composition of this disclosure is preferably an emulsified composition, and more preferably in a form in which an oil phase is dispersed in an aqueous phase (e.g., an oil-in-water (O / W) emulsion). In this case, in addition to at least one bio-extracted ceramide selected from soy sauce lees-extracted ceramide composition, Aspergillus oryzae-extracted ceramide composition, and chicken foot-extracted ceramide composition, it may contain oils and fats, nonionic surfactants, phospholipids, proteins, polysaccharides, pH adjusters, thickeners, fragrances, natural pigments, preservatives, and the like.

[0032] 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.

[0033] In this disclosure, the average particle size of dispersed particles means the volume-average particle size measured using the dynamic scattering method.

[0034] 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.).

[0035] 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: Dilute the sample separated from the ceramide dispersion composition of the present invention with pure water so that the concentration of ceramide contained in the sample is 0.1% by mass, and measure it 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. The volume-average particle size (Mv) is then determined.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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).

[0040] 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.

[0041] In this dispersion process, the dispersion treatment is preferably carried out by high-pressure emulsification treatment from the viewpoint of miniaturizing the dispersed particles.

[0042] 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.

[0043] 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.).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.).

[0052] 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.

[0053] 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.

[0054] When the ceramide composition disclosed herein is used in the pharmaceutical field (including pharmaceuticals and quasi-drugs), The composition (hereinafter sometimes referred to as "the pharmaceutical composition relating to this disclosure") may consist solely of each bio-extracted 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, the formulation form is not particularly limited, and the active ingredient and other components can be mixed by conventional methods to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewables, and soft tablets as oral compositions. For example, tablets can be manufactured by tableting. Both direct tableting, where the mixed raw materials are compressed directly, and granular tableting, where the mixed raw materials are granulated before tableting, can be used. Furthermore, for example, capsules can be either soft capsules or hard capsules. For example, external compositions can be used as creams, lotions, gels, etc. For example, compositions for intestinal administration can be used as foams.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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]

[0066] 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.

[0067] 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.

[0068] <Aspergillus oryzae-extracted ceramide composition> 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 this solid and mixed, then 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, then stirred and extracted overnight at 40°C. The process was carried out. The obtained 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 products were used as the koji mold-extracted ceramide composition.

[0069] <Chicken foot extract ceramide composition> Dried chicken was mixed with 2.5 times the volume of ethanol / water mixture and stirred overnight at 40°C for extraction. After collecting the extract, 1.2 times the volume of ethanol solution was added to the solid portion and mixed, and stirred overnight at 40°C for extraction. 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 products were used as the chicken foot extract ceramide composition.

[0070] 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.

[0071] 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.

[0072] [Table 1]

[0073] Furthermore, the free ceramide molecular species of the Aspergillus oryzae-extracted ceramide composition are shown in Table 2.

[0074] [Table 2]

[0075] Nano-sized ceramides Propylene glycol (other polyols may be added as needed), an emulsifier, and a ceramide composition were mixed and heated to 100-150°C (approximately the melting point of ceramide) until homogenized. Lecithin aqueous solution was then added and stirred, and the resulting mixture was processed under 150 MPa 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.

[0076] 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.

[0077] In the following, the soy sauce lees-extracted ceramide composition was nano-sized and used as the nano-sized ceramide composition.

[0078] 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. Chicken foot may be written as Avian. Maruzen Pharmaceutical Co., Ltd. (Pine Cera Powder) was used as glucosidoceramide, Takasago International Corporation (Ceramide NG "CERAMIDE TIC-001") was used as synthetic ceramide, and Fujifilm Wako (N-acetyl-L-cysteine) was used as NAC.

[0079] [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 inactivated. 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 (Dulbecco's Modified Eagle Medium; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, CA, USA). The preparation involved dissolving 4.75 g of Dulbecco's Modified Eagle Medium (DMEM) medium "Nissui" (2) (Nissui Pharmaceutical, Tokyo, Japan) in 470 mL of Milli-Q water, and adding 0.2 M L-glutamine ( Wako Pure Chemical Industries, Osaka, Japan: 10 mL, 50,000 U / mL penicillin; Meiji Seika Pharma, Tokyo, Japan: 1 mL, 0.05 mg / mL streptomycin; Meiji Seika Pharma: 0.5 mL The solution was prepared by adding 6 mL of 10% NaHCO3 (Fujifilm Wako Pure Chemical Industries).

[0080] Mitochondrial activity measurement using IN Cell Analyzer 2200 HaCaT cells 6.0 × 10 4 Cells were seeded in a 96-well plate at a density of cells / mL and pre-cultured for 24 hours. Then, ceramide was added to a final concentration of 5 μM. After 24 hours, the culture was performed. After removing the substrate, 100 μL / well of MitoTracker Red (Invitrogen, USA), diluted to 2.5 μM in 10% FBS / DMEM, was added to the plate and incubated at 37°C under 5% CO2 conditions for 30 minutes. After 1 minute, remove the medium containing MitoTracker Red, add 100 μL / well of MitoTracker Green (Invitrogen) diluted to 200 nM with 10% FBS / DMEM, and incubate for 30 minutes at 37°C under 5% CO2 conditions. A bait was prepared. Then, 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. Afterward, the diluted Hoechst 33345 solution was removed from the 96-well plate, 150 mL of PBS was added, and fluorescence was measured using an IN Cell Analyzer 2200 (GE Healthcare). Detection was performed. The fluorescence levels of Mito Tracker Red, Mito Tracker Green, and Hoechst 33342 were measured. By determining the activity, number, and area of ​​mitochondria within cells, we investigated these parameters. Images were analyzed using IN Cell Investigator High-content image analysis software (GE Healthcare).

[0081] 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 The cells were irradiated using UVB, and then immediately incubated in 10 mL of DMEM medium containing 10% FBS at 37°C in the presence of 5% CO2. Untreated cells were not irradiated with UVB. Only the cells were replaced. After 24 hours, the culture media of the non-UVB and UVB irradiated cells were discarded, washed with 1x PBS, the cells were detached with trypsin, and 3x10 cells were placed in a 96-well plate. 3 Sowing to form cells After 4 hours, the cells 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. The mixture was left undisturbed at room temperature for 30 minutes in the dark. Then, 5 μM BES-H2O2-AC in HEPES (pH 7.4) was added in a 1:1000 ratio. Hoechst 33342 (Dojindo) was mixed in a 1:400 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.

[0082] 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. 2 Using irradiated cells, the same experiment as described in "Measurement of Mitochondrial Activity Using IN Cell Analyzer 2200" was performed.

[0083] 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 then 1.5 mL of trypsin was added to permeate the entire dish and removed. The cells were then fertilized at 37°C under 5% CO2 conditions. After incubation for 1 minute, the cells were detached with DMEM, collected in a 15 mL tube, and centrifuged at 12000 rpm for 3 minutes to collect a cell pellet. LC-MS was used to analyze the components of the HaCaT cells. The fatty acid composition of ceramide was analyzed.

[0084] Evaluation of the effects of ceramides on HaCaT cells via Caco-2 cells: Barrier function in the intestinal tract Consideration > 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). The DMEM medium was prepared by mixing 470 mL of Milli-Q water with Dulbecco's Modified Eagle Medium (DMEM) medium "Nissui" (2) (manufactured by Nippon Suisan). Dissolve 4.75 g of the drug (Tokyo, Japan), and add 10 mL of 0.2 M L-glutamine (Fujifilm Wako Pure Chemical Industries, Osaka, Japan), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma, Tokyo, Japan), and 0.05 mg / mL streptomycin (Meiji Seika Pharma) 0.5 mL, 10% NaHCO3 (Fujifilm) (Wako Pure Chemical Industries) 6 mL was added and the mixture was prepared.

[0085] Mitochondrial activity measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells 1.0 × 10 2 Cells were seeded in a 24-well plate (Corning, NY, USA) at a concentration of cells / mL. After 24 hours, ceramide at a final concentration of 5 μM was added and incubated for another 24 hours. 6.0 × 10⁶ 4 96 wells at cells / mL 50 μL of the culture medium of HaCaT cells seeded on a plate and incubated for 24 hours was removed, and 50 μL of the culture medium of Caco-2 cells was added thereto. 48 hours later, the mitochondria of HaCaT cells were measured by the same method as described in the above "Measurement of Mitochondrial Activity Using IN Cell Analyzer 2200" The number and area, and the mitochondrial membrane potential activity were measured.

[0086] ROS measurement using IN Cell Analyzer 2200 (Caco-2 cells → HaCaT cells) Caco-2 cells were seeded in a 24-well plate (Corning, NY, USA) at 1.0×10 2 cells / mL. 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 "ROS Measurement Using IN Cell Analyzer 2200"

[0087] The UV damage recovery effect of ceramides in UVB-induced senescent cells (Caco-2 cells → HaCaT cells) Caco-2 cells were seeded in a 24-well plate (Corning, NY, USA) at 1.0×10 2 cells / mL. After 24 hours, ceramide at a final concentration of 5 μM was added and cultured for 24 hours.

[0088] HaCaT cells in a sub-confluent state were irradiated with UVB at 10 mJ / cm 2 and seeded in a 96-well plate at 6.0×10 4 cells / mL. After 24 hours, 50 μL of the culture medium of HaCaT cells was removed, and Caco-2 cells' culture medium 50 μL was added thereto. After 48 hours, "UV of Ceramide in UVB-Induced Aging Cells" The number and area of ​​mitochondria in HaCaT cells were measured using the same method as described in "Recovery Effect". The chondrial membrane potential activity was measured.

[0089] 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.

[0090] Millicell ERS-2 (Millipore Japan, Tokyo) was used to measure transepithelial electrical resistance. First, set the electrodes in the Millicell ERS-2 and sterilize them by immersing them in a 70% ethanol solution for 10 minutes. After disinfection, the electrodes were dried and then immersed in DMEM medium. Next, the Cell Culture Insert Electrodes were inserted on the side and outside, respectively, and measurements were taken. The measured values ​​were calculated using the following formula as a reference.

[0091] TER (Ω·cm 2 ) = (measured value - blank) × (insert area)

[0092] [Results of the review] Endogenous SIRT1 expression level (HaCaT cells) Endogenous SIRT1 expression levels were measured by quantitative RT-PCR after treating HaCaT cells with ceramide for 48 hours. As a result, SIRT1 expression was enhanced in HaCaT cells treated with glycosylceramide, soy sauce lees ceramide, Aspergillus oryzae ceramide, and Avian ceramide (Figure 1).

[0093] Endogenous SIRT3 expression level (HaCaT cells) Endogenous SIRT3 expression levels were measured by quantitative RT-PCR after treating HaCaT cells with ceramide for 48 hours. As a result, SIRT3 expression was enhanced in HaCaT cells treated with soy sauce lees ceramide, Aspergillus oryzae ceramide, Avian ceramide, nano-sized ceramide, and non-nano-sized ceramide (Figure 2).

[0094] Mitochondrial activity measurement using IN Cell Analyzer 2200 The number of mitochondria per cell, area (Mitotracker Green), and mitochondrial membrane potential activity (Mitotracker Red) of HaCaT cells treated with ceramide for 48 hours were measured using IN Cell Analyzer. Measurements were taken using a 2200. Glycosylceramide, synthetic ceramide, soy sauce lees ceramide, Aspergillus oryzae ceramide. Lamid, Avianceramide, and non-nano-sized ceramide significantly increased the number of mitochondria in HaCaT. Glycosylceramide, soy sauce lees ceramide, koji mold ceramide, and avian ceramide significantly increased the mitochondrial area of ​​HaCaT (Figure 3).

[0095] 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 4).

[0096] 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 treatment with ceramide for 48 hours (Figure 5).

[0097] Evaluation of human-type ceramide synthesis from natural human-type ceramides using LC-MS HaCaT cells from subconflu were treated with ceramide for 48 hours, and the adipocytes of the HaCaT cells were analyzed using LC-MS. The acid composition was measured. As a result, in HaCaT cells to which soy sauce lees ceramide, koji mold ceramide, and Avian ceramide were added, the amount of human-type ceramide with 24 carbon atoms and long-chain fatty acids significantly increased. (Figure 6).

[0098] Verification of endogenous SIRT1 expression levels (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 expression level of endogenous SIRT1 was measured by quantitative RT-PCR. As a result, SIRT1 was significantly enhanced in HaCaT treated with nano-sized ceramide (Figure 7).

[0099] Verification of endogenous SIRT3 expression levels (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 endogenous SIRT3 expression level was measured by quantitative RT-PCR. As a result, SIRT3 was significantly enhanced in HaCaT treated with Aspergillus oryzae ceramide (Figure 8).

[0100] 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 the IN Cell Analyzer 2200 (Figure 9).

[0101] 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. 2 HaCaT cells that have been irradiated with UVB ROS levels were measured using an IN Cell Analyzer 2200 after adding the substances to cells and treating them for 24 hours. As a result, Aspergillus oryzae ceramide and nano-sized ceramide significantly reduced ROS (Figure 10).

[0102] 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 HaCaT cells that have been irradiated with UVB 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 cells and treating them for 24 hours (Figure 11).

[0103] Evaluation of barrier function enhancement by natural human-type ceramides in CaCo-2 cells Measurement of endogenous SIRT1 expression levels (Caco-2 cells) The endogenous SIRT1 expression level was measured by quantitative RT-PCR after treating Caco-2 cells with ceramide for 24 hours. As a result, SIRT1 was significantly enhanced in Caco-2 cells to which synthetic ceramide, Avianceramide, and nano-sized ceramide were added (Figure 12).

[0104] 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 SIRT3 in Caco-2 cells treated with glycosylceramide and avianceramide (Figure 13a).

[0105] Measurement of endogenous ZO-1 expression levels (Caco-2 cells) Endogenous ZO-1 expression levels were measured by quantitative RT-PCR after treating Caco-2 cells with ceramide for 24 hours. As a result, ZO-1 expression was significantly enhanced in Caco-2 cells treated with soy sauce lees ceramide, Aspergillus oryzae ceramide, and nano-sized ceramide (Figure 13b).

[0106] Verification of Transepithelial Electrical Resistance (TER) (Caco-2 cells) Caco-2 cells seeded in cell inserts were treated with ceramide and differentiated for 14 days. Furthermore, TER values ​​were measured every 24 hours. As a result, soy sauce lees ceramide, koji mold ceramide, and Avia were found to have high TER values. In Caco-2 cells treated with ceramide, nano-ceramide, and non-nano-ceramide, the TER value significantly increased. Furthermore, during the first half of the 14-day measurement period (days 2-6), soy sauce lees ceramide and koji mold ceramide showed a significant increase in TER values. The TER values ​​of Caco-2 cells treated with lamidoceramide increased particularly significantly, and in the latter half of the study (days 7-144), the TER values ​​of Caco-2 cells treated with soy sauce lees ceramide and Avian ceramide also increased particularly significantly. (Figures 14a and 14b).

[0107] The sequences of the PCR primer sets (F and R: forward and reverse) used in the study are shown in the table below.

[0108] [Table 3]

Claims

1. A composition comprising a ceramide composition for use as a skin barrier, an intestinal barrier, or for anti-skin aging, The ceramide composition is at least one bio-extracted ceramide composition selected from the group consisting of soy sauce lees extract ceramide composition, Aspergillus oryzae extract ceramide composition, and chicken foot extract ceramide composition. composition.

2. The composition according to claim 1, which is for use as a skin barrier or for anti-skin aging.

3. The composition according to claim 2 for caring for skin damage caused by ultraviolet rays.

4. The composition according to claim 1, for use as an intestinal barrier.

5. The composition according to claim 4, for strengthening tight junctions in the intestinal tract.

6. The composition according to any one of claims 1 to 5, comprising at least t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h.

7. The total amount of t18:0-24:0h, t18:1-24:0h, and t20:0-24:0h is 30% by mass or more of the total amount of free ceramide contained. The composition according to claim 6.

8. The composition according to any one of claims 1 to 7, wherein the ceramide composition is an O / W type emulsion composition, and the volume-average particle size of the dispersed oil phase particles is 10 to 500 nm.

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