Method for extracting ceramide-containing material or ceramide from whole apples and / or apple pomace and composition containing the ceramide

Abstract

[Problem] A method for extracting a ceramide-containing material or ceramide from whole apples and / or apple juice extraction residue. Furthermore, to ensure utility as a food, especially a functional food, or a raw material for a cosmetic or medicine, that includes the ceramide-containing material. [Solution] In the present invention, an adsorbent is added to an ethanol treatment solution of whole apples and / or apple juice extraction residue, the mixture is stirred and subjected to solid-liquid separation, and solid material precipitated from the solution is separated, after which water is added to the resultant concentrate and the temperature thereof is maintained, whereby the resultant settled component is recovered and dried to obtain a ceramide-containing material or ceramide. Using the ceramide obtained from whole apples and / or apple juice extraction residue, there are formed a composition for promoting production of hyaluronic acid and / or type II collagen, and a composition for promoting improvement of memory impairment and / or enhancement of the level of phosphorylation of extracellular signal-related kinases (ERK1 / 2).

Description

[Technical Field]

[0001] The present invention (A) relates to a method for extracting apple-derived ceramide-containing material or apple-derived ceramide from whole apples and / or apple pomace, and further to a method for extracting ursolic acid (also called ursolic acid) and a polyphenol solution while fractionating them.

[0002] The present invention also relates to the use of ceramide as a food ingredient, particularly a functional food ingredient, or a cosmetic or pharmaceutical ingredient. More specifically, the present invention relates to a composition containing ceramide obtained from whole apples and / or apple pomace. It should be noted that "ceramide" and "ceramide-containing substance" as used herein are derived from apples. [Background technology]

[0003] Regardless of the variety, apples contain various useful components such as ceramides, ursolic acid, and polyphenols, as well as dietary fiber such as pectin. They are also rich in potassium, and it has been reported that when consumed as food, these components act to promote nutritional intake and ultimately have health-maintaining and disease-preventing effects.

[0004] In particular, in today's aging society, there is a demand to maintain beauty and health and improve the quality of life, and the above-mentioned problems can be solved by extracting components contained in apples and using them as ingredients for safe foods, particularly functional foods, or cosmetics or pharmaceuticals.

[0005] The production of apple juice generally involves using whole apples after harvest as raw materials, washing them, crushing them, and squeezing them to extract the juice components. Although the useful components contained in the original whole apples remain in the apple pomace, it is characterized by rapid spoilage and difficulty in storage, leading to problems such as the generation of industrial waste, air pollution, and environmental pollution, and is therefore disposed of as waste, requiring a lot of expense and effort. Extracting useful components from apple pomace and scientifically verifying their functionality could reduce waste disposal costs and potentially enable their use as ingredients in foods, particularly functional foods, cosmetics, or pharmaceuticals. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-57378 [Patent Document 2] Japanese Patent Application Publication No. 2019-154296 [Patent Document 3] Patent application No. 2020-24773 [Patent Document 4] Patent application No. 2020-510057

[0007] The following prior art documents are reported regarding the present invention (A). Patent Document 1 is a published application for a patent invention (B) filed earlier by the inventors of the present invention (A). Patent Document 1 discloses a pectin process in which whole apples and / or apple juice residue are used as raw materials, and are subjected to water treatment and drying in Common Step 1, followed by ethanol extraction in Common Step 2, thereby extracting pectin from the dried product after the ethanol treatment, and a ceramide process in which ceramide is extracted from the ethanol-treated solution. In contrast, the present invention (A) describes a method for extracting a ceramide-containing substance or ceramide from the ethanol-treated solution, a method for obtaining recovered ethanol, the effects of the ceramide-containing substance on cultured cells derived from skin or cartilage, and its industrial use, as well as a method for extracting a ursolic acid and / or polyphenol solution in the process for extracting the ceramide-containing substance. Patent Document 2 describes a method for efficiently concentrating apple-derived glucosylceramide by subjecting apples or their juice residue to heat treatment and enzyme treatment, but does not disclose or suggest a method for extracting ursolic acid and / or polyphenol solutions other than apple-derived ceramide. Patent Document 3 describes a method for producing a food product that utilizes the extraction of components derived from citrus peels and fruits by adding shochu or vinegar to the peels and fruits of citrus fruits. However, it does not disclose or suggest a method for extracting ceramide, ursolic acid, and / or polyphenol solution derived from apples, or the effect of ceramide-containing substances on dementia models. Patent Document 4 describes that animal experiments and / or experiments using cultured cells have confirmed that nicotine, nobiletin, sinesetin and / or cinpi are pharmaceutical compositions effective in treating central neurodegenerative diseases, but does not disclose or suggest a method for extracting apple-derived ceramide, ursolic acid and / or polyphenol solution and / or the effect of a ceramide-containing substance on a dementia model. [Non-patent literature]

[0008] [Non-Patent Document 1] "Functional Evaluation of Peach-Derived Ceramide" (Yuki Kobayashi et al.), FOOD STYLE 21, vol. 19, pp. 22-26 (2015). [Non-patent document 2] "Nobiletin, an anti-dementia component of Citrus Fruit Peel, suppresses the neurotoxic expression of amyloid beta peptide (Aβ) and improves Aβ-induced memory impairment" (Tetsu Yamakuni et al.), YAKUGAKU ZASSHI, vol. 130, pp. 517-520 (2010). [Non-patent document 3] T. Maurice, M. Hiramatsu, J. Itoh, T. Kameyama, T. Hasegawa, and T. Nabeshima, Brain Res., vol. 647, pp. 44-56 (1994). [Non-patent document 4] YJ. Huang, CH. Lina, HY. Lanea and GE Tsai, Current Neuropharmacology, vol. 10, pp. 272-285 (2012). [Non-Patent Document 5] JP Adams and JD Sweatt, Annu. Rev. Pharmacol. Toxicol., vol. 42, pp. 135-163 (2002).

[0009] Non-Patent Document 1 discloses the following about the effect of peach-derived ceramide on cultured human skin cells. Specifically, the hypothesis is first presented that adding peach-derived ceramide to cultured human skin cells increases the amount of ceramide contained in the cells, and thus transdermal application of ceramide improves the skin's moisturizing effect and barrier function. Furthermore, the following is also stated: Oral administration of peach-derived ceramide to humans reduced the amount of water evaporation from the skin, increased the moisture content of the stratum corneum, and improved skin texture. Therefore, oral administration, like transdermal administration, is expected to improve the skin's moisturizing effect and barrier function. Furthermore, like other plant-derived ceramides, peach-derived ceramide is composed of multiple ceramide components, and its sphingoid base is similar to that of rice, corn, or konjac, and / or its fatty acid composition is similar to that of apples. Ceramides derived from plants such as rice, corn, and konjac have all been shown to improve the moisturizing effect of skin when taken orally or administered transdermally, and a similar moisturizing effect was confirmed for peach-derived ceramides. Non-Patent Document 2 discloses nobiletin contained in citrus fruits as an example of a search for natural compounds with functions to prevent the onset of cognitive dysfunction and alleviate symptoms, following the elucidation of the pathological onset mechanism of cognitive dysfunction, the number of cases of which has been increasing in recent years. The apple-derived ceramide-containing substance disclosed in the present invention (A) is a different component from nobiletin derived from citrus fruits, and its mechanism of action is different from that of the dementia model used. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention (A) aims to provide a ceramide-containing substance or a method for extracting ceramide from whole apples and / or apple pomace. Furthermore, the invention discloses the effectiveness of the ceramide-containing substance on cultured cells derived from human skin and cartilage, and on neuronal model cells derived from dementia model mice and / or rats. The invention aims to utilize the substance as a food ingredient, particularly a health food ingredient, or as a cosmetic or pharmaceutical ingredient. [Means for solving the problem]

[0011] This invention (A) provides a method for extracting ceramide-containing substances or ceramide from whole apples and / or apple pomace. Furthermore, related to these, the invention discloses the effects of ceramide-containing substances on cultured cells derived from skin and cartilage, and on neural model cells derived from dementia model mice and / or rats. Additionally, the invention discloses methods for obtaining ursolic acid and / or polyphenol solutions, respectively, and recovering and reusing ethanol in the extraction method. More details are as follows.

[0012] Figure 1 shows the process for extracting a ceramide-containing substance from whole apples and / or apple juice pomace as product 1. The common process in Figure 1 refers to the pretreatment process common to the ceramide process and pectin process disclosed in the prior patent invention (B). In Figure 1, a box surrounded by a thin line indicates a process, and a box surrounded by a thick line indicates a substance. In other words, the process of obtaining an ethanol-treated solution and a dried product after ethanol treatment (common process 1 and / or common process 2, Figure 1) through common process 1 in which whole apples and / or apple juice pomace are treated with water and then dried, and common process 2 in which ethanol is added to the water-treated dried product for extraction and solid-liquid separation is carried out, results in (A) a step of adding an adsorbent to the ethanol-treated solution, stirring the mixture, and removing the adsorbent by solid-liquid separation to obtain an adsorbent-treated solution (ceramide step 1, FIG. 1 and / or FIG. 9); (B) a step of leaving the adsorbent-treated liquid to stand and removing the precipitated solid matter by solid-liquid separation to obtain a precipitate-free liquid (ceramide step 2, FIG. 1 and / or FIG. 10); (C) concentrating the precipitate-removed solution under reduced pressure to obtain a concentrated solution and recover ethanol (ceramide step 3, FIG. 1 and / or FIG. 11); (D) A process of adding water to the concentrated liquid, incubating the liquid, and separating the precipitated components into solid and liquid, followed by drying to obtain a ceramide-containing substance as a solid component (ceramide process 4, FIG. 1 and / or FIG. 12).

[0013] In addition, in the present invention (A), the effect of the ceramide-containing substance extracted as described above on cultured cells derived from skin and cartilage is as follows: (E) The effect of ceramide-containing products on the production of hyaluronic acid in cultured cells derived from human skin (Fig. 2 and / or Fig. 3). (F) The effect of ceramide-containing materials on the production of type II collagen in cultured cells derived from human cartilage (FIG. 4 and / or FIG. 5) is disclosed.

[0014] Furthermore, in the present invention (A), the effect of the ceramide-containing substance extracted as described above on neural model cells derived from dementia model mice and / or rats was investigated. (G) The effect of ceramide-containing products on dementia model mice with induced memory impairment (Figure 6). (H) The effect of ceramide-containing substances on the regulation of neural signal transmission in rat-derived neural model cells (FIG. 7 and / or FIG. 8) is disclosed.

[0015] Furthermore, in the present invention (A), in each of the ceramide step 1 to ceramide step 4, (I) In the ceramide step 2, the solid matter that precipitates when the adsorbent-treated solution is allowed to stand is ursolic acid (ceramide step 2, FIG. 9). (J) In the ceramide step 3, the precipitate-removed solution can be concentrated under reduced pressure to recover and reuse ethanol (ceramide step 3, FIG. 10). (K) In the ceramide step 4, the ceramide-containing material precipitates when water is added to the concentrated solution and kept warm, and the resulting solution obtained by solid-liquid separation is a polyphenol solution (ceramide step 4, FIG. 11).

[0016] In addition, the present invention (A) discloses (L) separating and purifying ceramide from the ceramide-containing material extracted by the ceramide steps 1 to 4 (FIG. 12).

[0017] Figure 14 shows the entire process for extracting ceramide-containing material from whole apples and / or apple pomace as Product 1, the intermediate steps of obtaining ursolic acid as Product 2 from Ceramide Step 2, recovered ethanol from Ceramide Step 3, and a polyphenol solution as Product 3 from Ceramide Step 4, and illustrates the entire process for obtaining ceramide from the ceramide-containing material. Ceramide Steps 1 to 4 are multi-step processes, but are characterized by the fact that products are obtained by separating them from each step. In Figure 14, the products after separation are shown uniformly with the liquid component on the left and the solid component on the right.

[0018] As used herein, "whole apples" refers to all or any of the harvested apples that maintain their original form as apples after harvesting, regardless of variety or freshness, including apples for fresh consumption or processing that are distributed in the market or are being distributed before distribution, as well as apples that have not yet reached the stage of processing such as apple juicing. Even apples at intermediate stages before harvest can include apple-forming bodies that retain the original form or components of an apple. In other words, "whole apples" refers to all harvested apples that retain the original form as apples, such as fallen apples, unripe apples, and apples rejected during the sorting process.

[0019] As used herein, apple pomace refers to the solid portion obtained by separating the liquid portion from apples using a known method, such as crushing, grinding, and / or squeezing, or the like, or simply crushed or ground apples. Specifically, as one example, apples are crushed to a size of approximately 5 to 30 mm using a hammer crusher or the like, and juice is extracted at a pressure of approximately 5 kg / cm to 20 kg / cm. However, because the condition of apples varies greatly depending on the variety, there are no particular restrictions on the degree of crushing or squeezing. In addition to crushing (crushing to a size of approximately 5 to 30 mm using a hammer crusher or the like) and grinding (crushing to a size of approximately 5 mm or less using a colloid mill or pulper or the like), disintegration (loosening aggregated solids using a mesh, screen, or the like) may also be performed. In addition to the above, in this specification, apple pomace includes each and every "pomace" obtained from the juicing process at apple juicing factories, but also includes so-called apple processing by-products that are generated during various processing processes at juicing factories, juice factories, processing factories, and other factories, including peels and cores that remain after cutting in the process of producing cut apples.

[0020] Furthermore, the term "ceramide" in this specification does not simply refer to ceramide as a compound in which sphingosine and fatty acid are bound by an amide bond, but refers to glucosylceramide or galactosylceramide in which glucose or galactose is bound to ceramide.These ceramides are also called cerebrosides.Ceramides derived from plants such as apples exist in the form of cerebrosides, but since the name "ceramide" is generally established, this term will also be used in this application.In other words, the term "ceramide" referred to in this specification refers to cerebroside in which glucose or galactose is bound to ceramide as a compound in which sphingosine and fatty acid are bound by an amide bond.

[0021] The hyaluronic acid whose production is enhanced in this specification is a type of polysaccharide called mucopolysaccharide present in animals, and is generally widely distributed in connective tissues such as the vitreous body of the eye, the umbilical cord, joint synovial fluid, vascular lining, and gastric mucosa. It is water-soluble and has high water retention, and its aqueous solution is viscous.

[0022] Additionally, the collagen whose production is enhanced in this specification is a type of protein present in animals, and is a fibrous component present in bones, cartilage, tendons, skin, etc. Type II collagen is mainly present in cartilage.

[0023] The N-methyl-D-aspartate receptor (NMDA receptor) whose inhibition is reversed in this specification is a receptor present in animal nerve cells that is involved in memory, learning, neuronal cell death, etc. It has been reported that inhibition of this receptor causes memory impairment seen in dementia such as Alzheimer's disease (see Non-Patent Documents 3 and 4 cited above).

[0024] Furthermore, the ERKs referred to herein as enhanced are extracellular signal-regulated kinases 1 / 2, which are signal transduction factors widely present in living organisms. When cells receive external signals, ERK1 / 2 are phosphorylated and activated. In neurons, ERK1 / 2 is known to be involved in memory formation, and activation of ERK1 / 2 can lead to the alleviation and / or treatment of dementia symptoms (see Non-Patent Document 5 cited above).

[0025] In addition, MEK (MAPK / ERK Kinase) enhanced in this specification is an enzyme that phosphorylates ERK1 / 2. The MEK / ERK signaling system is a signaling pathway that acts upstream of ERK1 / 2 and has been shown to play an important role in transducing extracellular signals into cellular responses. In neurons, MEK and ERK1 / 2 are known to be involved in memory formation, leading to the alleviation and / or treatment of dementia symptoms (see Non-Patent Document 5 cited above).

[0026] Furthermore, in this specification, ursolic acid (also referred to as ursolic acid) is a five-membered triterpene carboxylic acid found in plants, and refers to a compound found in various trees, particularly in the waxy substance of their fruits and / or leaves.

[0027] Additionally, in this specification, polyphenols refer to pigment compounds and / or bitter components that are widely distributed in plants. In the case of apples, multiple polyphenol compounds are present. Pigment compounds are compounds that make up the pigments of apple skin and / or fruit, and are anthocyanins and / or their glycosides, with the types of compounds that make up different apple varieties. The main polyphenol component of the red pigment found in apple skin is cyanidin-3-glucoside.

[0028] Apples also contain polyphenols called procyanidins, which are bitter-tasting compounds and exist as a mixture of polymers of catechins.

[0029] This invention (A) discloses each of the steps from Ceramide Step 1 to Ceramide Step 4, which extract ceramide-containing substances, useful substances contained in apples, from whole apples and / or apple pomace. Furthermore, in Ceramide Steps 2 to 4, the steps of extracting ursolic acid and / or polyphenol solutions are described. Ceramide and / or ursolic acid and / or polyphenols are collective terms for known compounds and / or compounds that are widely distributed in apples and other plants, respectively, and the unique feature of apples is that they contain all of these useful components. Furthermore, this invention (A) makes it possible to extract these useful substances from raw materials or residues that are industrially generated in large quantities, such as whole apples and / or apple pomace. [Effects of the Invention]

[0030] This invention (A) provides a means or method for extracting a ceramide-containing substance from whole apples and / or apple pomace. It also discloses that the ceramide-containing substance has an effect on cultured cells derived from human skin and cartilage. Furthermore, in the process of extracting the ceramide-containing substance, a solution of ursolic acid and polyphenols is obtained, and its effects are generally as follows: (Effect 1) This invention (A) makes it possible to extract ceramide-containing substances as Result 1 from whole apples and / or apple juice residue. By implementing this invention (A), it becomes possible to effectively utilize unused resources that pollute the environment with putrid odors in apple juice factories, and to reduce the cost of disposing of residues that are currently treated as waste.

[0031] (Effect 2) In the present invention (A), when a ceramide-containing substance is added to cultured cells derived from human skin, the amount of hyaluronic acid produced by the cells increases, and at the same time, the gene expression of the enzyme that synthesizes hyaluronic acid is enhanced.

[0032] (Effect 3) In the present invention (A), when a ceramide-containing substance is added to cultured cells derived from human cartilage, the amount of type II collagen produced by the cells increases, and the expression of genes that produce type II collagen is enhanced.

[0033] (Effect 4) The composition containing ceramide or a ceramide-containing substance of the present invention (A) improves memory impairment and / or enhances phosphorylation of ERK, a signal transduction factor.

[0034] (Effect 5) In the present invention (A), by adding an adsorbent to the ethanol-treated solution obtained by ethanol extraction of whole apples and / or apple pomace, it is possible to decolorize the solution, remove odorous components, and precipitate and separate ursolic acid as product 2.

[0035] (Effect 6) In the present invention (A), an ethanol-treated solution of whole apples and / or apple juice residue is treated with an adsorbent, and the precipitated ursolic acid is removed from the solution, which is then concentrated under reduced pressure to obtain a concentrated solution containing ceramide, and the ethanol can be recovered and reused.

[0036] (Effect 7) In the present invention (A), by adding water to the concentrated solution and keeping it warm, ceramide-containing substances are removed as solids, and a polyphenol solution can be extracted as product 3 into the solution.

[0037] (Effect 8) In the present invention (A), ceramide can be separated and purified from the ceramide-containing material.

[0038] (Effects of the Invention (A) by Claim) According to claim 1, a ceramide-containing substance can be extracted as product 1 from an ethanol treatment solution of whole apples and / or apple pomace. According to claim 2, the addition of a ceramide-containing substance extracted from an ethanol-treated solution of whole apples and / or apple pomace can enhance the production of hyaluronic acid in human cells, as well as the production of type II collagen in human cells. According to claim 3, a ceramide-containing substance can be extracted from an ethanol-treated solution of whole apples and / or apple pomace, which can either improve memory impairment or increase the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2). According to claim 4, ursolic acid can be extracted as product 2 from the ethanol treatment solution of whole apples and / or apple pomace. According to claim 5, ethanol can be recovered and reused from the ethanol treatment solution of whole apples and / or apple juice residue. According to claim 6, a polyphenol solution can be extracted as the result 3 from an ethanol treatment solution of whole apples and / or apple pomace. According to claim 7, ceramide can be separated and purified from a ceramide-containing substance extracted from an ethanol-treated solution of whole apples and / or apple pomace. According to claim 8, the ceramide-containing substance and / or ursolic acid and / or polyphenol solution extracted from the ethanol-treated solution of whole apples and / or apple pomace can be used as ingredients for food and / or cosmetics, respectively. According to claim 9, a composition for promoting the production of hyaluronic acid and / or type II collagen by administration can be obtained. According to claim 10, a composition can be obtained that promotes improvement of memory impairment and / or an increase in the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2) by administration. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows a process for extracting a ceramide-containing substance as product 1 from whole apples and / or apple pomace. [Figure 2] This shows the effect of ceramide-containing products on the production of hyaluronic acid by cultured human skin-derived cells. [Figure 3] This shows the effect of ceramide-containing products on the expression of genes that produce hyaluronic acid in cultured cells derived from human skin. [Figure 4] 1 shows the effect of ceramide-containing materials on the production of type II collagen by cultured human cartilage-derived cells. [Figure 5] 1 shows the effect of ceramide-containing products on the expression of genes that produce type II collagen in cultured human cartilage-derived cells. [Figure 6] 1 shows the effect of a ceramide-containing product on memory impairment caused by the NMDA receptor blocker MK-801. [Figure 7] 1 shows the effect of ceramide-containing products on the phosphorylation of ERK1 / 2 in rat-derived neuronal model cells. [Figure 8] 1 shows the effect of ceramide-containing products on the MEK / ERK signaling system in rat-derived neuronal model cells. [Figure 9] Ceramide step 1: This shows the adsorbent treatment step of the ethanol-treated solution. [Figure 10] Ceramide process 2: This shows the process of extracting ursolic acid as precipitate removal solution and product 2 from the adsorbent-treated solution. [Figure 11]Ceramide step 3: This shows the step of obtaining a concentrated solution and recovered ethanol from the precipitate-removed solution. [Figure 12] Ceramide Process 4: This shows the process of extracting a ceramide-containing substance as Product 1 and a polyphenol solution as Product 3 from the concentrated solution. [Figure 13] Separation and purification of ceramide: This shows the process for obtaining ceramide from the ceramide-containing product of Product 1. [Figure 14] The entire process for extracting the various useful components from whole apples and / or apple pomace is shown below. DETAILED DESCRIPTION OF THE INVENTION

[0040] Common Step 1, in which water is added to whole apples and / or apple pomace, followed by stirring and drying, and Common Step 2, in which ethanol is added to the water-treated and dried whole apples and / or apple pomace, followed by stirring and solid-liquid separation to separate into an ethanol-treated liquid and a post-ethanol-treated dried product, are the same as those described in the prior patent invention (B) (see Patent No. 5902256 relating to Patent Document 1). A mode for carrying out the present invention (A) comprises the following steps. While these steps are carried out in order, each step is independent, and it is possible to repeat each step before proceeding to the next. In other words, the present invention (A) comprises the following steps. 1. Ceramide step 1, in which an adsorbent is added to the ethanol-treated liquid obtained in the common step 2 and stirred, and then the adsorbent is removed by solid-liquid separation, while an adsorbent-treated liquid is obtained. 2. A ceramide step 2 in which the adsorbent-treated liquid is allowed to stand, and the precipitated solid matter is removed by solid-liquid separation. 3. Ceramide step 3, in which the precipitate treatment solution is concentrated under reduced pressure to obtain a concentrated solution and recover ethanol. 4. Ceramide step 4, in which water is added to the concentrated solution, followed by incubating the solution to obtain a precipitate, which is then separated into solid and liquid and dried to extract a ceramide-containing substance as product 1.

[0041] Furthermore, the present invention (A) provides the following: 5. The effect on the amount of hyaluronic acid produced by cultured human skin fibroblasts when the ceramide-containing material extracted as Product 1 from the ceramide process 4 is dissolved in a solvent and added to the culture medium. And / or, the effect on the amount of expression of genes involved in hyaluronic acid production when genes (RNA) are extracted from the cells and comprehensive gene expression analysis and real-time PCR analysis using primers are performed.

[0042] 6. The effect on the amount of type II collagen produced by cells when the ceramide-containing material extracted as Product 1 from the ceramide process 4 is dissolved in a solvent and added to a culture medium of human chondrosarcoma cells. And / or the effect on the amount of expression of genes involved in the production of type II collagen when genes (RNA) are extracted from the cells and analyzed by real-time PCR using primers.

[0043] 7. As demonstrated by the dementia model mice described below, administration of a solution in which the Result 1 from the ceramide step 4 is suspended results in recovery from memory impairment.

[0044] 8. As demonstrated by rat-derived neural model cells described below, when the ceramide-containing substance extracted as Result 1 from the ceramide process 4 is dissolved in a solvent and administered, it has the effect of activating the phosphorylation of factors involved in the cell signaling pathway, and the effect of recovering from memory impairment is due to this pathway.

[0045] 9. A method for extracting ursolic acid as product 2 in the ceramide step 2 by allowing the adsorbent-treated liquid to stand and subjecting the precipitated solid to solid-liquid separation and drying. 10. A method in which the precipitate-removed solution in the ceramide step 3 is concentrated under reduced pressure to obtain recovered ethanol. 11. A method for extracting a polyphenol solution as product 3 from the solution portion obtained by adding water to the concentrated liquid in the ceramide process 4 and then incubating the precipitate obtained by solid-liquid separation. 12. A method for separating and purifying ceramide from the ceramide-containing material extracted as product 1 in the ceramide step. [Example]

[0046] In the following, Example 1 will be described a process for extracting a ceramide-containing substance as Result 1 from an ethanol-treated solution of the water-treated and dried product of whole apples and / or apple pomace.

[0047] (Ceramide step 1) Addition of adsorbent to ethanol-treated solution (Figures 1, 9, and / or 14) An adsorbent is added to the alcohol-treated solution of the water-treated and dried product obtained in common process 1 (Figure 1 and / or Figure 14), and the mixture is stirred. The liquid and adsorbent are then separated by solid-liquid separation. This process is referred to as ceramide process 1. Examples of adsorbents include activated carbon and activated clay. The amount of adsorbent added is 100 to 150 g, preferably 120 g, per kg of the initial water-treated and dried residue. The stirring time is preferably approximately 10 to 20 minutes, and more preferably approximately 10 minutes. The stirring temperature is between 40 and 70°C, but stirring is preferably performed at 50 to 60°C. After stirring, the liquid and adsorbent are separated by centrifugation or suction filtration. The adsorbent is discarded. The liquid after filtration is left to stand overnight at a temperature of 5 to 15°C.

[0048] (Ceramide step 2) A step of removing solid matter precipitated from the adsorbent-treated solution (FIG. 1, FIG. 10, and / or FIG. 14). In the above process, the adsorbent-treated solution obtained from the adsorbent treatment is left standing overnight, and the solution contains precipitates. Therefore, the liquid and precipitates are separated by solid-liquid separation methods such as centrifugation or suction filtration, preferably by suction filtration using analytical filter paper. The precipitate-removed solution obtained is used in the next step.

[0049] (Ceramide step 3) Preparation of concentrated solution by vacuum concentration of precipitate-removed solution (FIG. 1, FIG. 11, and / or FIG. 14) The precipitate-removed liquid obtained in the ceramide process 2 is concentrated under reduced pressure to obtain a concentrated liquid. This is referred to as the ceramide process 3. The concentration method is performed using a concentration means such as a centrifugal thin-film vacuum evaporator or a rotary evaporator. The concentration is preferably performed under reduced pressure by heating at a temperature below the boiling point. The device required for the pressure reduction is a pressure reduction means such as a diaphragm pump. Other pressure reduction methods can also be used as long as they can maintain the required degree of pressure reduction (approximately 0.1 atmospheres) or vacuum.

[0050] (Ceramide step 4) Add water to the concentrated solution and incubate to extract ceramide-containing material as product 1 (Figure 1, Figure 12, and / or Figure 14). Water is added to the concentrate obtained in ceramide step 3 and kept warm, resulting in solid-liquid separation of the precipitated solid component and the aqueous solution. This is referred to as ceramide step 4. The amount of water added is 3 to 5 volume parts, preferably 4 volume parts, per volume part of the concentrate. The stirring time is preferably approximately 10 to 50 minutes, with 30 minutes being particularly desirable. The stirring temperature is between 40 and 70°C, but stirring is preferably performed at 50 to 60°C. Since the solid component precipitates in the stirred aqueous solution, the solid component is separated by solid-liquid separation, preferably suction filtration. The resulting solid component is then washed with warm water and dried under reduced pressure in a desiccator. The solid component referred to here is the ceramide-containing component of product 1. The yield from 1 kg of the original water treatment and drying residue is 1.5 to 2.0%.

[0051] As disclosed in Example 1, the water-treated and dried product was used as a raw material, and ceramide-containing material was extracted as Result 1 by carrying out ceramide steps 1 to 4, with the results shown below. (1) In the ceramide step 1, an adsorbent was added to the ethanol-treated liquid, and after stirring, an adsorbent-treated liquid was obtained. (2) In the ceramide step 2, solid matter precipitated from the adsorbent-treated solution was removed to obtain a precipitate-removed solution. (3) In the ceramide step 3, the precipitate-removed liquid was concentrated under reduced pressure to obtain a concentrated liquid. (4) In the ceramide step 4, the concentrated liquid was added with water and kept warm to obtain a ceramide-containing substance as product 1. The yield per 1 kg of residue was 15 to 20 g. [Example]

[0052] In Example 2, the effects of ceramides obtained from whole apples and / or apple pomace on human skin and cartilage are disclosed using as an example a ceramide-containing material extracted as Product 1 after ceramide steps 1 to 4 of Example 1.

[0053] (Effect of ceramide-containing substances on hyaluronic acid production by cultured human skin cells) (Figure 2) Human fibroblasts (HSF) were cultured in D-MEM medium supplemented with 10% bovine serum. The ceramide-containing material extracted through steps 1 to 4 was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at concentrations of 1 μg / mL, 5 μg / mL, and 20 μg / mL. The cells were then cultured for 72 hours. After culture, the supernatant was collected, and the amount of hyaluronic acid produced was quantified and evaluated by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 2. The horizontal axis of the figure represents the concentration of the ceramide-containing material added, and the vertical axis represents the amount of hyaluronic acid produced. Addition of the ceramide-containing material at concentrations of 0.1 μg / mL and 5 μL increased the amount of hyaluronic acid produced, and a significant difference was observed when 5 μg / mL of the ceramide-containing material was added compared to the control.

[0054] (Comprehensive analysis of gene expression in cultured human skin cells containing ceramide) Human fibroblasts (HSF) were cultured in D-MEM medium supplemented with 10% bovine serum. The ceramide-containing material extracted through steps 1-4 was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at 5 μg / mL and 20 μg / mL. After 24 hours, total RNA was extracted and comprehensively analyzed for the expression of skin-beautifying genes using next-generation sequencing. While type I and type III collagen, which are abundant in skin, tended to increase, the increase was only 1.3- to 1.5-fold, and no significant increase was observed. On the other hand, the hyaluronic acid synthase HAS1 showed a significant increase of 8.8-fold with the addition of 5 μg / mL of the ceramide-containing material and 11.4-fold with the addition of 20 μg / mL. Furthermore, the hyaluronic acid-degrading enzyme HYAL1 decreased by 0.8-fold with the addition of 5 μg / mL of the ceramide-containing material and 0.5-fold with the addition of 20 μg / mL.

[0055] (Effect of ceramide-containing foods on the expression of hyaluronic acid-producing genes) (Figure 3) Human fibroblasts (HSF) were cultured in D-MEM medium supplemented with 10% bovine serum. The ceramide-containing material extracted in step 4 above was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at 5 μg / mL and 20 μg / mL. Expression of the hyaluronan synthase gene, HAS1, was confirmed by real-time PCR. The results are shown in Figure 3. The horizontal axis represents the concentration of the ceramide-containing material added, and the vertical axis represents the ratio of expression of the hyaluronan-producing gene, HAS1, to expression of GAPDH, a housekeeping gene commonly expressed at a constant level throughout the cells. Addition of 5 μg / mL of the ceramide-containing material resulted in approximately two-fold increase in HAS1 expression compared to the control, and addition of 20 μg / mL resulted in approximately three-fold increase in HAS1 expression, confirming the enhanced expression observed in the RNA sequencing analysis.

[0056] (Effect of ceramide-containing materials on the production of type II collagen by cultured human cartilage-derived cells) (Figure 4) Human chondrosarcoma cells (OUMS27) were cultured in D-MEM medium supplemented with 10% bovine serum. The ceramide-containing material extracted in the ceramide step 4 was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at 1.0 μg / mL, 5 μg / mL, and / or 20 μg / mL. The cells were then cultured for 72 hours. After culture, the supernatant was collected, and the amount of type II collagen produced was quantified and evaluated by ELISA. The results are shown in Figure 4. The horizontal axis of the figure represents the concentration of the ceramide-containing material added, and the vertical axis represents the amount of type II collagen produced. The amount of type II collagen produced tended to increase as the ceramide-containing material was added to 1.0 μg / mL, 5 μg / mL, and / or 20 μg / mL.

[0057] (Effect of ceramide-containing products on gene expression in cultured human cartilage-derived cells) (Figure 5) Figure 5 shows the results of analyzing gene expression of COL2A1, a type II collagen production gene, when a ceramide-containing substance extracted by the ceramide step 4 described above was added to the culture medium of human chondrosarcoma cells (OUMS27). The horizontal axis of the figure represents the concentration of the added ceramide-containing substance, and the vertical axis represents the ratio of the expression level of the type II collagen production gene COL2A1 to the expression level of GAPDH, a housekeeping gene, i.e., a gene commonly expressed at a constant level in cells. When 1.0 μg / mL of the "ceramide-containing substance" was added, COL2A1 gene expression was increased by approximately 2.0-fold, and at 5 μg / mL, it was increased by approximately 2.7-fold.

[0058] The effects of the ceramide-containing material disclosed in Example 2 on cultured human skin-derived cells and / or cartilage-derived cells can be summarized as follows. (1) Ceramide-containing products induced the expression of HAS1, a gene involved in the production of hyaluronic acid, in human skin fibroblasts, thereby enhancing the production of hyaluronic acid. (2) Ceramide-containing materials increased the expression of COL2A1, a gene involved in the production of type II collagen, in human chondrocytes, resulting in increased production of type II collagen. Therefore, according to the present invention, a composition for achieving this purpose can be formulated using ceramide obtained from whole apples and / or apple pomace as an active ingredient for promoting the production of hyaluronic acid and / or type II collagen. Note that the above points can be applied not only to Result 1 of Example 1 but also to apple-derived ceramides in general. [Example]

[0059] In Example 3, the effect of ceramide obtained from whole apples and / or apple pomace on improving memory impairment and / or increasing the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2) is disclosed using as an example a ceramide-containing product extracted as Product 1 through the ceramide steps 1 to 4 of Example 1.

[0060] (Effect of ceramide-containing foods on memory impairment caused by the N-methyl-D-aspartate receptor blocker MK-801) (Figure 6) N-methyl-D-aspartate (NMDA) receptors, a type of glutamate receptor, are important for memory formation, and administration of the non-competitive NMDA receptor blocker MK-801 to mice causes memory impairment. In this example, the effects of ceramide-containing substances on memory impairment were evaluated by a passive avoidance test using dementia model mice administered with MK-801. Seven-week-old male ddY mice (Japan SLC Co., Ltd.) were bred and then used in the experiment. Ceramide-containing compounds were suspended in 0.5% sodium carboxymethylcellulose (CMC) solution and orally administered to the mice at doses of 100 mg / kg body weight or 500 mg / kg body weight once daily for 6 days. A passive avoidance training trial was conducted on the 7th day after the start of administration, and a retention trial was conducted on the 8th day. On the 7th day, 90 minutes before the passive avoidance training trial, mice were orally administered 100 mg / kg body weight or 500 mg / kg body weight of ceramide-containing compounds or the vehicle (0.5% CMC solution). Sixty minutes later, the noncompetitive NMDA receptor blocker MK-801 (0.2 mg / kg body weight) or saline was subcutaneously administered. Thirty minutes later, a passive avoidance training trial was conducted, followed by a retention trial 24 hours later. The passive avoidance test was performed using a step-through passive avoidance response apparatus. This apparatus consisted of a lighted compartment and a dark compartment where an electrical stimulus was applied. First, in a training trial, the mouse was placed in the lighted compartment, and upon entering the dark compartment, an electrical stimulus (0.4 mA, 1 second) was administered. Next, in a retention trial, the mouse was placed in the lighted compartment again 24 hours later, and the time it took to enter the dark compartment (response latency) was measured and used as an index of memory. Here, the training trial refers to the process of placing a mouse in the lighted compartment and teaching it to remember that it will receive an electrical stimulus if it enters the dark compartment. The retention trial refers to the process of placing the mouse in the lighted compartment again 24 hours after the training trial and measuring the response latency until it enters the dark compartment. When the mouse remembers that it will receive an electrical stimulus in the dark compartment, the response latency in the retention trial becomes longer. When the mouse is administered the NMDA receptor blocker MK-801, memory impairment is induced, the response latency becomes shorter, and the mouse moves to the dark compartment. In the passive avoidance test, this response latency is used as an index to evaluate the effect of improving memory impairment. The test results are shown in Figure 6. In the retention trial, the response latency was significantly reduced in the MK-801 alone group compared to the vehicle-administered control group, confirming memory impairment. On the other hand, the response latency was significantly prolonged in the 100 mg / kg body weight group administered the ceramide-containing product compared to the MK-801 alone group, demonstrating that MK-801-induced memory impairment was improved in the 100 mg / kg body weight group administered the ceramide-containing product.

[0061] (Effect of ceramide-containing products on the phosphorylation of extracellular signal-regulated kinase (ERK1 / 2) in neuronal model cells) (Figure 7) PC12 cells, derived from a rat adrenal pheochromocytoma, are widely used as a neural model cell. PC12 cells were cultured in D-MEM medium supplemented with 5% bovine serum and 5% horse serum. A ceramide-containing compound dissolved in dimethyl sulfoxide (DMSO) was added to the cells at a concentration of 30 μg / mL and cultured for 5, 10, 30, and 60 minutes. After culture, the cells were harvested, and the levels of phosphorylated ERK1 / 2 (phospho-ERK1 / 2), which is important for memory formation, were assessed by Western blotting using anti-phospho-ERK1 / 2 and / or anti-total ERK1 / 2 antibodies. The results are shown in Figure 7. Treatment with the ceramide-containing compound resulted in an increase in the phosphorylation level of ERK1 / 2, peaking 10 minutes after treatment, indicating activation of ERK1 / 2.

[0062] (Effect of ceramide-containing products on the MEK / ERK signaling pathway in neural model cells) (Figure 8) The effect of ceramide-containing products on the MEK / ERK signaling pathway in PC12 cells was evaluated using an inhibitor of MEK, an upstream kinase of extracellular signal-regulated kinase (ERK1 / 2). PC12 cells were cultured in D-MEM medium containing 5% bovine serum and 5% horse serum. The MEK inhibitor U0126 was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at a concentration of 10 μM. Thirty minutes later, ceramide-containing products dissolved in DMSO were added to the cells at a concentration of 30 μg / mL and incubated for 10 minutes. After incubation, cells were harvested, and the levels of phosphorylated ERK1 / 2 (phospho-ERK1 / 2), which is important for memory formation, were assessed by Western blotting using anti-phospho-ERK1 / 2 and / or anti-total ERK1 / 2 antibodies. The results are shown in Figure 8. A sample treated with nobiletin (30 μM) was used as a positive control. Treatment with ceramide-containing products increased the phosphorylation level of ERK1 / 2, but this increase was suppressed by pretreatment with the MEK inhibitor U0126, confirming that ceramide-containing products activate the MEK / ERK signaling pathway.

[0063] The effects of the ceramide-containing substance on dementia model mice and / or neuronal model cells disclosed in Example 3 can be summarized as follows. (1) Ceramide-containing products improved memory impairment in dementia model mice administered MK-801, an N-methyl-D-aspartate (NMDA) receptor blocker. (2) In PC12 cells, a neuronal model cell, ceramide-containing materials increased the phosphorylation level of extracellular signal-regulated kinases (ERK1 / 2), which are important for memory formation, thereby activating ERK1 / 2. Therefore, according to the present invention, a composition for achieving the purpose of improving memory impairment and / or promoting an increase in the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2) can be formulated using ceramide obtained from whole apples and / or apple pomace as an active ingredient. Note that the above points can be applied not only to Result 1 of Example 1 but also to apple-derived ceramides in general. [Example]

[0064] In Example 4, ursolic acid and / or recovered ethanol, which are product 2 and / or polyphenol solution, which are obtained in the intermediate steps from the ceramide step 2 to the ceramide step 4, are disclosed.

[0065] (Ceramide Process 2) A process for extracting ursolic acid as product 2 from the adsorbent-treated solution (FIG. 1, FIG. 10, and / or FIG. 14). The adsorbent-treated solution obtained from the adsorbent treatment in the ceramide step 1 was left standing overnight to form a solution containing precipitates, so product 2 was extracted by solid-liquid separation methods such as centrifugation or suction filtration, preferably suction filtration using analytical filter paper. The yield of product 2 from 1 kg of the original water treatment and drying residue was 0.9 to 1.5%.

[0066] (Analysis of ursolic acid by thin layer chromatography) Product 2 extracted in the ceramide step 2 was dissolved in a 4:1 mixture of chloroform and methanol, and applied to a thin silica gel layer. Thin layer chromatography was performed using a 65:25:4 mixture of chloroform, methanol, and water as the developing solvent. After development, a copper-phosphate color reagent was sprayed onto the surface of the silica gel layer and heated to develop color. As a result, product 2 extracted as a precipitate in the ceramide step 2 was confirmed to be ursolic acid.

[0067] (Ceramide step 3) Recovery of ethanol by vacuum concentration of the precipitate-removed solution obtained in ceramide step 2 (FIG. 1, FIG. 11, and / or FIG. 14). The precipitate-removed liquid obtained in the ceramide process 2 is concentrated under reduced pressure to obtain a concentrated liquid and recovered ethanol, which can be reused. The recovered ethanol can be reused as ethanol in the ceramide process 1. In a typical example, the ethanol recovery rate was 70%. Furthermore, when the water content of the recovered ethanol was measured using a hydrometer, it was found to maintain the original water content, making it possible to reuse the ethanol without the need for an additional process.

[0068] (Ceramide Step 4) Preparation of Product 3 Polyphenol Solution by Adding Water to the Concentrate and Incubating (Figures 1, 12, and / or 14) In the ceramide step 4, water is added to the concentrate obtained in the ceramide step 3 and kept warm, and the precipitated solid component and aqueous solution are separated into solid and liquid components. The liquid portion is a polyphenol-containing substance. In a typical example, 2 L of water (four times the volume) is added to 0.5 L of the concentrate, and the resulting precipitate is washed with another 2 L of water. A total of 4.5 L of filtrate is obtained as a polyphenol solution.

[0069] (Analysis of polyphenol solution obtained in ceramide step 4) In the ceramide step 4, after adding water and incubating, the ceramide-containing precipitate is separated into a solid and liquid solution, resulting in a polyphenol solution. The polyphenol content of this solution was measured by the Folin-Denis method using catechin as the standard substance, and the amount of polyphenols contained in 100 mL of solution was calculated to be 150 mg in terms of catechin. Therefore, the concentration of the polyphenol solution was 0.15%, and 6.75 g was obtained from 1 kg of the initial water treatment and drying residue.

[0070] As disclosed in Example 4, the results obtained in each step from ceramide step 2 to ceramide step 4 are as follows: (1) In the ceramide step 2, ursolic acid was extracted from the adsorbent-removed solution as product 2. The concentration was 0.9% to 1.5% per kg of the original water treatment and dried product. (2) In the ceramide process 3, it was possible to prepare a concentrated solution and recover ethanol. The recovery rate of ethanol was 70%. (3) In the ceramide step 4, the concentrated solution was diluted with water and kept warm to remove the precipitated solid components, yielding a polyphenol solution. The volume obtained from 1 kg of residue was 4.5 L, and the polyphenol concentration was 150 mg / 100 mL in terms of catechin. [Example]

[0071] In Example 5, the separation and purification of the ceramide-containing material extracted in the ceramide step 4 and instrumental analysis are disclosed.

[0072] (Separation and purification of ceramide-containing substances by silica gel column chromatography) (Figures 13 and 14) 0.2 g of the ceramide-containing material extracted in the ceramide extraction step 4 was dissolved in 2 mL of a 4:1 mixture of chloroform and methanol. Next, a 30 mm diameter glass column was filled with silica gel (preferably 230 to 400 mesh, manufactured by Merck) suspended in a 4:1 mixture of chloroform and methanol to a height of 420 mm. The column volume was approximately 300 mL. The solution containing the ceramide-containing material was adsorbed onto the tip of the silica gel column, and a 4:1 mixture of chloroform and methanol was passed down at a flow rate of 2 mL / min. The solution flowing out from the bottom of the column was dispensed into test tubes numbered 1 to 40 in 10 mL increments. Test tubes 15 to 34 were spotted onto a silica gel thin-layer plate (Merck, silica gel 60F254, 10 cm x 20 cm) and developed using a developing solvent consisting of a 65:25:4 mixture of chloroform, methanol, and water. After development, anthrone sulfate reagent was sprayed onto the plate and heated to develop color. Commercially available ceramide preparations (derived from soybeans) were spotted on both ends of the thin-layer plate as standard substances, and the mobility and color were compared. Ceramide was separated into test tubes 31 to 33 by column chromatography of the ceramide-containing material.

[0073] (Separation and purification of ceramide by preparative high-performance liquid chromatography) (Figure 13 and / or Figure 14) The samples from test tubes 31 to 33 separated by silica gel column chromatography were pooled and concentrated under reduced pressure, then dissolved in a solvent containing a 95:5 mixture of methanol and water. Preparative high-performance liquid chromatography was performed as follows: A reverse-phase chromatography column, preferably a TSKGELODS-80TS (Tosoh Corporation) with a column size of 8 mm in diameter and 300 mm in length, was connected to a high-performance liquid chromatography system, and a 95:5 mixture of methanol and water was used as the eluent. Elution was performed at a flow rate of 2 mL / min, and detection was performed at 210 nm. The component eluted at a retention time of 19.96 minutes was collected, concentrated to dryness, and subjected to instrumental analysis.

[0074] (Infrared absorption spectroscopy analysis of ceramide) The infrared absorption spectrum of the component eluted at 19.96 minutes from the preparative high-performance liquid chromatography was measured. The infrared absorption spectrum was measured by adjusting the sample using the KBr-disk method and measuring the transmittance (T%) from 4000 cm to 400 cm using a Fourier transform infrared spectrometer. This spectrum was consistent with ceramide.

[0075] (Analysis of ceramide by nuclear magnetic resonance spectroscopy) The component eluted at 19.96 minutes in the preparative high-performance liquid chromatography was dissolved in a 4:1 mixture of deuterated chloroform and deuterated methanol, and the nuclear magnetic resonance spectrum was measured using a 270 MHz Fourier transform nuclear magnetic resonance spectrometer (manufactured by JEOL Ltd.) with tetramethylsilane as an internal standard. The spectrum was consistent with that of ceramide.

[0076] The results of the separation and purification and / or instrumental analysis of the ceramide contained in the ceramide-containing material disclosed in Example 5 are summarized as follows. (1) Ceramide was isolated and purified from the ceramide-containing material by silica gel column chromatography followed by preparative high performance liquid chromatography. (2) The structure of the ceramide separated and purified by the above chromatography was analyzed by infrared absorption spectroscopy and / or nuclear magnetic resonance spectroscopy, which were consistent with that of ceramide. [Industrial Applicability]

[0077] This invention (A) provides a method for extracting a ceramide-containing substance or a ceramide and / or ursolic acid and / or polyphenol solution from the ethanol-treated solution obtained by treating whole apples and / or apple pomace with water and then drying them. This process makes it possible to utilize the components contained in the ethanol-treated solution. Furthermore, the ethanol used for extraction can be recovered and reused. This invention (A) not only makes it possible to use whole apples, which would otherwise be discarded without being eaten raw or processed, as a raw material, but also leads to the industrial use of apple pomace, which would otherwise be discarded as an unused resource or place a burden on the environment.

[0078] In this invention (A), it has been demonstrated at the substance and gene expression levels that the ceramide-containing material extracted in each of the above steps enhances the production of hyaluronic acid by cultured human skin cells and type II collagen by cultured human chondrocytes. Human hyaluronic acid and type II collagen each play important roles in human tissues and are substances that have a direct effect on maintaining health and beauty and improving quality of life. This is the first time that components contained in whole apples and apple pomace or apple juice pomace have been found to enhance the production of hyaluronic acid or type II collagen. Whole apples and apple pomace or apple juice pomace are available in large quantities, and the useful components obtained in each of the steps shown in Figures 1 to 14 above can be used industrially as ingredients in foods and cosmetics.

[0079] In this invention (A), the ceramide-containing substance extracted in each of the above steps improved memory impairment in a mouse dementia model and increased the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2), which is important for memory formation, in rat-derived neuronal model cells, i.e., activated ERK1 / 2. As the number of patients increases in an aging society, improving dementia or memory impairment has a direct impact on maintaining health and improving quality of life. This is the first time that components contained in whole apples and apple juice pomace have been found to improve dementia or memory impairment. Whole apples and apple juice pomace are available in large quantities, and the useful components obtained in each of the steps shown in Figures 1 to 14 above can be used industrially as ingredients in health foods and pharmaceuticals.

Claims

1. a ceramide step 1 in which water is added to whole apples and / or apple pomace and stirred for a water treatment, followed by drying to obtain a sample, ethanol is added to this sample and stirred to separate it into an ethanol-treated liquid and a post-ethanol-treated dried product, activated carbon or activated clay is added as an adsorbent to the ethanol-treated liquid obtained in this way, and the liquid is stirred, followed by removing the adsorbent by solid-liquid separation to obtain an adsorbent-treated liquid; a ceramide step 2 in which the adsorbent-treated solution is allowed to stand, thereby separating the precipitate that precipitates, thereby obtaining a precipitate-removed solution; a ceramide step 3 in which the precipitate-removed solution obtained from the ceramide step 2 is concentrated under reduced pressure to obtain a concentrated solution; a ceramide step 4 in which water is added to the concentrated solution obtained in the ceramide step 3, the resulting solution is kept warm, and the precipitated solid component is separated and dried to obtain a ceramide-containing substance as a result 1; A method for extracting a ceramide-containing substance, comprising:

2. The method for extracting a ceramide-containing substance according to claim 1, wherein the ceramide-containing substance obtained as product 1 after passing through each of the ceramide steps 1 to 4 is one or both of the following: a substance that significantly increases the production of hyaluronic acid upon administration; and a substance that significantly increases the production of type II collagen.

3. The method for extracting a ceramide-containing substance according to claim 1, wherein the ceramide-containing substance obtained as product 1 after passing through each of the ceramide steps 1 to 4 improves memory impairment upon administration and / or causes an increase in the phosphorylation level of extracellular signal-regulated kinase (ERK1 / 2).

4. The method for extracting a ceramide-containing substance according to any one of claims 1 to 3, wherein ursolic acid is obtained as a precipitate as a product 2 in the ceramide step 2.

5. 5. The method for extracting a ceramide-containing substance according to claim 1, wherein ethanol is recovered in the ceramide step 3 and reused as ethanol in the ceramide step 1.

6. The method for extracting a ceramide-containing substance according to any one of claims 1 to 5, wherein a polyphenol solution is obtained as a resultant product 3 in the ceramide step 4.

7. a ceramide step 1 in which water is added to whole apples and / or apple pomace and stirred for a water treatment, followed by drying to obtain a sample, ethanol is added to this sample and stirred to separate it into an ethanol-treated liquid and a post-ethanol-treated dried product, activated carbon or activated clay is added as an adsorbent to the ethanol-treated liquid obtained in this way, and the liquid is stirred, followed by removing the adsorbent by solid-liquid separation to obtain an adsorbent-treated liquid; a ceramide step 2 in which the adsorbent-treated solution is allowed to stand, thereby separating the precipitate that precipitates, thereby obtaining a precipitate-removed solution; a ceramide step 3 in which the precipitate-removed solution obtained from the ceramide step 2 is concentrated under reduced pressure to obtain a concentrated solution; a ceramide step 4 in which water is added to the concentrated solution obtained in the ceramide step 3, the resulting solution is kept warm, and the precipitated solid component is separated and dried to obtain a ceramide-containing substance as a result 1; obtaining ceramide from the ceramide-containing material; A method for extracting ceramide, comprising:

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