METHOD FOR EXTRACTING CERAMIDE-CONTAINING MATERIAL OR CERAMIDE FROM WHOLE APPLES AND/OR APPLE JUICE EXTRACTION RESIDUE, AND COMPOSITION INCLUDING SAID CERAMIDE

Abstract

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).

Claims

1. A method for extracting ceramide-containing matter comprising: a ceramide step 1 wherein whole apples or apple juice extraction residues are treated with water and then dried to obtain a sample, then the sample is added with ethanol and stirred for separation into an ethanol treatment liquid and post-ethanol treatment dried matter, and the resulting ethanol treatment liquid is added and stirred with an adsorbent, after which the adsorbent is removed by solid-liquid separation to obtain an adsorbent treatment liquid, a ceramide step 2 wherein deposits precipitating by letting the adsorbent treatment liquid standing are separated to obtain a precipitate-free liquid, a ceramide step 3 wherein the precipitate-free liquid obtained from the ceramide step 2 is concentrated under reduced pressures to obtain a concentrate, and a ceramide step 4 wherein a solid ingredient settling down by adding water to the concentrate obtained from the ceramide step 3 followed by heat insulation is separated and dried to obtain ceramide-containing matter as Product 1.

2. A method for extracting ceramide-containing matter as recited in claim 1, wherein, upon administration, the ceramide-containing matter obtained as Product 1 by way of the respective ceramide steps 1 through 4 brings about a significant rise of production of hyaluronic acid or a significant rise of production of type II collagen.

3. A method for extracting as recited in claim 1, wherein, upon administration, the ceramide-containing matter obtained as Product 1 by way of the respective ceramide steps 1 through 4 contributes to an improvement of memory impairment or a rise of phosphorylation level of extracellular signal-regulated kinase (ERK1/2).

4. A method for extracting ceramide-containing matter as recited in claim 1, wherein ursolic acid is precipitated as Product 2 in the ceramide step 2.

5. A method for extracting ceramide-containing matter as recited in claim 1, wherein ethanol is collected in the ceramide step 3 for reuse as the ethanol in the ceramide step 1.

6. A method for extracting ceramide-containing matter as recited in claim 1, wherein a poly-phenol solution is obtained as Product 3 in the ceramide step 4.

7. A method for extracting ceramide comprising: a ceramide step 1 wherein whole apples or apple juice extraction residues are treated with water and then dried to obtain a sample, then the sample is added with ethanol and stirred for separation into an ethanol treatment liquid and post-ethanol treatment dried matter, and the resulting ethanol treatment liquid is added and stirred with an adsorbent, after which the adsorbent is removed by solid-liquid separation to obtain an adsorbent treatment liquid, a ceramide step 2 wherein deposits precipitating by letting the adsorbent treatment liquid standing are separated to obtain a deposit-free liquid, a ceramide step 3 wherein the precipitate-free liquid obtained from the ceramide step 2 is concentrated under reduced pressures to obtain a concentrate, a ceramide step 4 wherein a solid ingredient settling down by adding water to the concentrate obtained from the ceramide step 3 followed by heat insulation is separated and dried to obtain ceramide-containing matter as Product 1, and a step of obtaining ceramide from the ceramide-containing matter.

8. Foodstuffs, or a cosmetic material containing the ceramide-containing matter obtained by the method for extracting ceramide set forth in claim 1.

9. A composition for facilitation of hyaluronic acid and/or type II collagen, which contains ceramide obtained from whole apples or apple juice extraction residues.

10. A composition for facilitation of an improvement of memory impairment or a rise of phosphorylation level of extracellular signal-regulated kinase (ERK1/2), which contains ceramide obtained from whole apples or apple juice extraction residues.

11. Foodstuffs, or a cosmetic material containing ursolic acid obtained by the method for extracting ceramide set forth in claim 4.

12. Foodstuffs, or a cosmetic material containing the polyphenol solution obtained by the method for extracting ceramide set forth in claim 6.

Description

BRIEF EXPLANATION OF THE DRAWINGS

[0063] FIG. 1 depicts a stepwise method/process of extracting ceramide-containing matter as Product I from whole apples and/or apple juice extraction residues.

[0064] FIG. 2 depicts the effects of ceramide-containing matter on the production of hyaluronic acid by cultured cells derived from the human skin.

[0065] FIG. 3 depicts the effects of ceramide-containing matter on the expression of genes that produce hyaluronic acid by cultured cells derived from the human skin.

[0066] FIG. 4 depicts the effects of ceramide-containing matter on the production of type II collagen by cultured cells derived from the human cartilage.

[0067] FIG. 5 depicts the effects of ceramide-containing matter on the expression of genes that produce type II collagen by cultured cells derived from the human cartilage.

[0068] FIG. 6 depicts the effects of ceramide-containing matter on a memory impairment caused by NMDA receptor blocker MK-801.

[0069] FIG. 7 depicts the effects of ceramide-containing matter on the phosphorylation of ERK1/2 in neural model cells derived from rats.

[0070] FIG. 8 depicts the effects of ceramide-containing matter on the MEK/ERK signaling system in neural model cells derived from rats.

[0071] FIG. 9 depicts ceramide step 1 of treating an ethanol treatment liquid with an adsorbent.

[0072] FIG. 10 depicts ceramide step 2 of extracting ursolic acid a precipitate-free liquid and ursolic acid as Product 2 from an adsorbent-treated liquid.

[0073] FIG. 11 depicts ceramide step 3 of obtaining a concentrate and recovering ethanol from the precipitate-free liquid.

[0074] FIG. 12 depicts ceramide step 4 of extracting ceramide-containing matter as Product 1 and a polyphenol solution as Product 3 from the concentrate.

[0075] FIG. 13 depicts a step of obtaining ceramide from Product 1 or ceramide-containing matter by separation and purification.

[0076] FIG. 14 depicts a whole method/process of extracting such various useful ingredients as mentioned above from whole apples and/or apple juice extraction residues.

MODES FOR CARRYING OUT THE INVENTION

[0077] Common step 1 in which whole apples and/or apple juice extraction residues are stirred with the addition of water followed by drying, and common step 2 in which ethanol is added to the whole apples and/or apple juice extraction residues subjected to water treatment and drying, and they are then stirred together for solid-liquid separation thereby fractionating them into an ethanol treatment liquid and post-ethanol treatment dried matter are similar to those set forth in the prior invention (see Japanese Patent No. 5902256 corresponding to Patent Document 1; Japanese Patent Document 2015-57378A). The method/process mode for carrying out the present invention comprises steps as mentioned below. These steps are carried out in order; however, each independent step may be repeated and then go to the next step. More specifically, the method/process according to the present invention comprises the following steps.

Ceramide Step 1

[0078] An adsorbent is added to and stirred with the ethanol treatment liquid obtained in the aforesaid common step 2, after which the adsorbent is removed by solid-liquid separation to obtain the adsorbent treatment liquid.

Ceramide Step 2

[0079] Solid matter precipitating by letting the aforesaid adsorbent treatment liquid standing is removed by solid-liquid separation.

Ceramide Step 3

[0080] The aforesaid precipitate treatment liquid is concentrated under reduced pressures to obtain a precipitate and recover ethanol.

Ceramide Step 4

[0081] A precipitate obtained by adding water to the concentrate and subjecting the concentrate to heat insulation is subjected to solid-liquid separation and then dried to extract ceramide-containing matter as Product 1.

[0082] Further, the following effects or advantages are obtained from the present invention:

5. Effect on the amount of hyaluronic acid produced by cells in the case where the ceramide-containing matter extracted as Product 1 in the aforesaid ceramide step 4 and dissolved in a solvent is added to a culture medium of human skin fibroblasts cells, and/or Effect on the amount of expression of genes taking part in production of hyaluronic acid as found by extracting genes (RNAs) from cells for comprehensive analysis of gene expression and real-time PCR analysis using a primer.
6. Effect on the amount of type II collagen produced by cells in the case where the ceramide-containing matter extracted as Product 1 in the aforesaid ceramide step 4 and dissolved in a solvent is added to a culture medium of human cartilage sarcoma cells, and/or Effect on the amount of expression of genes taking part in human cartilage sarcoma cell production as found by extracting genes (RNAs) from cells for comprehensive analysis of gene expression and real-time PCR analysis using a primer.
7. Effect on recovery from memory impairment by administration of a solution wherein Product I from the aforesaid ceramide step 4 is suspended, as demonstrated by the dementia model mice to be described later.
8. Effect on the activation of phosphorylation of a factor taking part in a cellular signaling pathway in the case where the ceramide-containing matter extracted as Product 1 in the aforesaid ceramide step 4 is dissolved in a solvent for administration, and Effect of the aforesaid signaling pathway on the aforesaid recovery from memory impairment.
9. Method/process where in the aforesaid ceramide step 2, solid matter precipitating upon the adsorbent-treated liquid let standing is subjected to solid-liquid separation and dried thereby extracting ursolic acid as Product 2.
10. Method/Process where in the aforesaid ceramide step 3, the precipitate-free liquid can be concentrated under reduced pressures for recovery of ethanol.
11. Method/Process wherein in the aforesaid ceramide step 4, a polyphenol solution is extracted as Product 3 in a solution portion in which a precipitate obtained by adding water to the concentrate followed by heat insulation (heat-retaining) is subjected to solid-liquid separation.
12. Method/Process in which ceramide is separated and purified from ceramide-containing matter extracted as Product 1 in the aforesaid ceramide step.

EXAMPLE 1

[0083] The method/process step of extracting the ceramide-containing matter as Product 1 from an ethanol treatment liquid of a material obtained by water treatment and drying of whole apples and/or apple juice extraction residues will now be explained.

Ceramide Step 1: Addition of an Adsorbent to the Ethanol Treatment Liquid (FIGS. 1, 9 and/or 14)

[0084] The adsorbent is added to and stirred with an alcohol treatment liquid of water-treated/dried material obtained in common step 1 (FIGS. 1 and/or 14), followed by separation into a liquid portion and the adsorbent by solid-liquid separation. This is called ceramide step 1. The adsorbent used herein may be active carbon, activate clay and so on. The amount of the adsorbent to be added may be 100 grams to 150 grams, preferably 120 grams per 1 kg of the initial residues upon water treatment/drying. The stirring time may be about 10 to 20 minutes, most preferably about 10 minutes at a stirring temperature between 40° C. and 70° C., preferably between 50° C. and 60° C. After stirring, a liquid portion is separated from the adsorbent as by centrifugation or suction filtration. This adsorbent is thrown away. The post-filtration liquid portion is let standing for one night while kept at 5° C. to 15° C.

Ceramide Step 2: Removal of Solid Matter Precipitating from the Adsorbent Treatment Liquid (FIGS. 1, 10 and/or 14)

[0085] Referring to a solution obtained by letting the adsorbent treatment liquid obtained from the aforesaid adsorbent treatment standing for one night, it is separated into a liquid portion and precipitates by a solid-liquid separation method such as centrifugation or suction filtration, preferably suction filtration using an analytical filter paper, because the precipitates settle down. The precipitate-free liquid obtained as a solution will be used in the next step.

Ceramide Step 3: Preparation of a Concentrate by Concentration of the Precipitate-free Liquid Under Reduced Pressures (FIGS. 1, 11 and/or 14

[0086] A concentrate is obtained by concentration under reduced pressures of the precipitate-free liquid obtained in ceramide step 2. This is called Ceramide Step 3 in which concentration is carried out with concentration means such as centrifugal thin-film vacuum evaporation equipment or a rotary evaporator, preferably under concentration conditions comprising heating at reduced pressures and temperatures no higher than the boiling point. The equipment needed for reduced pressures makes use of reduced pressure means such a diaphragm pump. If the necessary degree of reduced pressures (about 0.1 atm.) or the necessary degree of vacuum can be maintained, other evacuation means may also be used.

Ceramide Step 4: Extraction of Ceramide-containing Matter as Product 1 by Adding Water to the Concentrate and Heat Insulation (Heat-retaining) (FIGS. 1, 12 and/or 14)

[0087] With water added to the concentrate obtained in ceramide step 3 and heat insulation, solid-liquid separation is carried out to separate a settling solid ingredient from an aqueous solution. This is herein called ceramide step 4. Water is added in an amount of 3 to 5 parts by volume, preferably 4 parts by volume per 1 part by volume of the concentrate. The stirring time is preferably about 10 to 50 minutes, more preferably 30 minutes, and the stirring temperature may be between 40° C. and 70° C., but preference is given to 50° C. to 60° C. The stirred aqueous solution, because of the solid component settling down therein, is subjected to solid-liquid separation, preferably suction filtration, for separation of the solid ingredient. The obtained solid ingredient is washed with warm water, and then dried under reduced pressure by desiccators. The solid ingredient referred to herein is Product I that is the ceramide-containing matter obtained in a yield of 1.5% to 2.0% as calculated on the basis of 1 kg of the original water-treated/dried residues.

[0088] Set out below are the results of extraction of the ceramide-containing matter as Product 1 by using the water-treated/dried matter as a starting material and carrying out ceramide steps 1 through 4.

(1) In ceramide step 1, the adsorbent was added to the ethanol-treated liquid followed by stirring, yielding an adsorbent-treated liquid.
(2) In ceramide step 2, the solid matter precipitating from the adsorbent-treated liquid was removed, yielding a precipitate-free liquid.
(3) In ceramide step 3, the precipitate-free liquid was concentrated under reduced pressure, yielding a concentrate.
(4) In ceramide step 4, water was added to the concentrate followed by heat insulation, yielding the ceramide-containing matter as Product 1 in a yield of 15 to 20 grams per 1 Kg of the residues.

EXAMPLE 2

[0089] In Example 2, the effects of ceramide obtained from whole apples and/or apple juice extraction residues are shown with reference to the ceramide-containing matter extracted as Product 1 by way of ceramide steps 1 through 4 in Example 1.

Effect of the Ceramide-containing Matter on the Production of Hyaluronic Acid by Human Skin-derived Cultured Cells (FIG. 2)

[0090] Human fibroblasts (HSF) were cultured in a D-MEM culture medium containing 10% bobbin serum. The ceramide-containing matter extracted by way of the foregoing ceramide steps 1 through 4 was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was added to the cells in such an amount as to give 1 μg/mL, 5 μg/mL and/or 20 μg/mL for 72-hour culture, after which the supernatant liquid was recovered to quantify and estimate the amount of production of hyaluronic acid by the ELISA method, and the results are plotted in FIG. 2 with the concentration of the added ceramide-containing matter as abscissa and the amount of production of hyaluronic acid as ordinate. As the ceramide-containing matter was added in such an amount as to give 0.1 μg/mL and/or 5 μg/mL, it caused the amount of production of hyaluronic acid to increase so that when 5 μg/mL of the ceramide-containing matter were added, there was a significant difference found from the control.

Comprehensive Analysis of the Ceramide-containing Matter Taking Part in Gene Expression by the Human Skin-deprived Cultured Cell

[0091] Human fibroblasts (HSF) were cultured in a D-MEM culture medium containing 10% bobbin serum. The ceramide-containing matter extracted by way of the foregoing ceramide steps 1 through 4 was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was added to the cells in such an amount as to give 5 μg/mL and/or 20 μg/mL. Twenty-four (24) hours after addition, all RNAs were extracted to use the next-generation sequencer for comprehensive analysis of transcription products by way of RNA sequence analysis of genes relating to skin care. As a result, type I collagen and type III collagen present abundantly in the skin had tended to increase, but not one of them increased significantly by a factor of 1.3 folds to 1.5 folds. On the other hand, when the ceramide-containing matter was added to HAS1 or a hyaluronic acid synthesis enzyme in an amount of 5 μg/mL and 20 μg/mL, there was a profound increase by a factor of 8.8 folds and 11.4 folds, respectively, and when the ceramide-containing matter was added HYAL1 or a hyaluronic acid-breaking enzyme in an amount of 5 μg/mL and 20 μg/mL, there was a decrease by a factor of 0.8 and 0.5 folds respectively.

Effect of the Ceramide-containing Matter on the Expression of Hyaluronic Acid Production Genes (FIG. 3)

[0092] Human fibroblasts (HSF) were cultured in a D-MEM culture medium containing 10% bobbin serum. The ceramide-containing matter extracted in the foregoing ceramide step 4 was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was added to the cells in such an amount as to give 5 μg/mL and/or 20 μg/mL to identify the expression of HAS1 or a hyaluronic acid synthesis enzyme gene by real-time PCR. The results are plotted in FIG. 3 with the concentration of the ceramide-containing matter added as abscissa and the amount of expression of hyaluronic acid production gene HAS1 as ordinate in term of a ratio to the amount of expression of GAPDH that were genes expressing in a constant amount commonly in housekeeping genes or cells. It has been understood that with reference to the cells with 5 μg/mL and 20 μg/mL of the ceramide-containing matter added thereto, respectively, the amount of expression of HAS1 is about two folds and about three folds more than the control, meaning that expression increases as is the case with the RNA sequence analysis.

Effect of the Ceramide-containing Matter on the Production of Type II Collagen by the Human Cartilage Derived Cultured Cell (FIG. 4)

[0093] The human cartilage sarcoma cell (OUMS27) was cultured in a D-MEM culture medium containing 10% bobbin serum. The ceramide-containing matter extracted in the foregoing ceramide step 4 was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was added to the cells in such an amount as to 1.0 μg/mL, 5 μg/mL and/or 20 μg/mL for 72-hour culture. After culture, the supernatant was recovered to quantify and estimate the amount of type II collagen produced by the ELISA method. The results are plotted in FIG. 4 with the amount of the ceramide-containing matter as abscissa and the amount of production of type II collagen. As the amount of the ceramide-containing matter increased from 1.0 μg/mL to 5 μg/mL and/or to 20 μg/mL, there was a tendency for the amount of production of type II collagen to increase.

Effect of the Ceramide-containing Matter on Gene Expression by the Human Cartilage-derived Cultured Cells (FIG. 5)

[0094] Upon the addition of the ceramide-containing matter extracted in the foregoing ceramide step 4 to a cultured solution of the human cartilage sarcoma cell (OUMS27), the results of gene expression of COL2A1 or type II collagen production genes are plotted in FIG. 5 with the concentration of the ceramide-containing matter as abscissa and the amount of expression of the type II collagen production genes as ordinate in terms of a ratio to the amount of expression of genes GAPDH expressing in a constant amount commonly in housekeeping genes or cells. In the cells added with the “ceramide-containing matter in an amount of 1.0 μg/mL and 5 μg/mL, respectively, the amount of expression of COL2A1 genes was almost doubled in the former and increased about 2.7 times in the latter.

[0095] The effects of the ceramide-containing matter set forth in Example 2 on the cultured cells derived from the human skin and/or cultured cells derived from the human cartilage are summarized below.

(1) The ceramide-containing matter induced the expression of hyaulronic acid production genes HAS1 in the cultured cells derived from the human skin fibroblasts to increase the production of hyaulronic acid.
(2) The ceramide-containing matter enhanced the expression of type II collagen production genes COL2A1 in the human cartilage cells, resulting in an increase in an amount of type II collagen production.

[0096] According to the present invention, therefore, it is possible to provide a composition capable of achieving such objects by using the ceramide obtainable from the whole apples and/or apple juice extraction residues as an effective ingredient for promotion or acceleration of hyaluronic acid and/or type II collagen. This may be applied not only to Product I of Example 1 but also to overall ceramides derived from apples.

EXAMPLE 3

[0097] In Example 3, the effects of the ceramide obtained from whole apples and/or apple juice extraction residues on improvement of memory impairments and/or influences on an increase in the phosphorylation level of extracellular signal-regulated kinase (ERK1/2) are clarified typically with reference to the ceramide-containing matter extracted as Product 1 by way of ceramide steps 1 through 4.

Effect of the Ceramide-containing Matter on Memory Impairments Caused by N-methyl-D-aspartate Receptor Blocker MK-801 (FIG. 6)

[0098] N-methyl-D-aspartate (NMDA) receptor that is one of glutamic acid receptors is important for memory formation; administration of MK-801 that is a non-competitive NMDA receptor blocker to mice gives rise to memory impairments.

[0099] In this example, passive avoidance tests using dementia model mice having MK-801 administered thereto were carried out for estimation of effects of the ceramide-containing matter on memory impairments.

[0100] More specifically, 7 week old male DDY mice (available from Japan SLC, Inc.) were pre-fed for experimentation. The ceramide-containing matter suspended in an aqueous solution of 0.5% carboxymethyl cellulose sodium (CMC) was orally administrated to the mice once per day in a dose of 100 mg/kg body weight or 500 mg/kg body weight for six days. At the 7.sup.th and 8.sup.th days from the onset of administration, training and retention trials of passive avoidance testing were carried out. Ninety (90) minutes before the training trial of passive avoidance testing, 100 mg/kg body weight or 500 mg/kg body weight of the ceramide-containing matter or an aqueous solution of solvent 0.5% CMC were orally administrated to the mice, followed by subcutaneous administration of non-competitive NMDA receptor blocker MK-801 (0.2 mg/kg body weight) or a physiological saline solution 60-minute later. Thirty (30) minutes after that, training trial of passive avoidance testing was carried out, and after the elapse of 24 hours, retention testing was carried out.

[0101] The passive avoidance testing was carried out using Step-through type passive avoidance reaction equipment made up of a lighting room and a dark room through which electrical stimulation runs. First of all, mice were allowed to come in the lighting room as training trial, after which an electrical stimulation (0.4 mA, one second) was provided to them when the mice entered the dark room. Twenty-four (24) hours after that, the mice were again allowed to come in the lighting room as retention trial to measure the time until they entered the dark room (reaction latency) as a memory index.

[0102] The training trial used herein is understood to stand for a method/process in which the mice were allowed to come in the lighting room and then enter the dark room where they were allowed to receive an electrical stimulation. The retention trial used herein is understood to stand for a method/process of measuring a time taken to allow the mice to again enter the lighting room 24 hours after training trial and then the dark room as reaction latency. When the mice have a memory of receiving an electrical stimulation in the dark room, the reaction latency becomes long upon retention trial. Administration of the NMDA receptor blocker MK-801 to the mice induces a memory impairment to make the reaction latency so short that they move to the dark room. In the passive avoidance testing, the effect on an improvement of memory impairment is estimated with the reaction latency as an index.

[0103] The test results are shown in FIG. 6. In the retention trial, a group to which MK-801 alone was administered was significantly lower in the reaction latency than a control group to which the solvent was administered, indicative of memory impairment. On the other hand, a group to which 100 mg/kg body weight were administered was significantly longer in reaction latency than a group to which MK-801 alone was administered; the group to which the ceramide-containing matter was administered in a dose of 100 mg/kg body weight was clearly improved in terms of memory impairment induced by MK-801.

Effect on the Phosphorylation of Extracellular Signal-regulated Kinase (ERK1/2) in Neural Model Cells (FIG. 7)

[0104] PC12 cells derived from rat adrenal gland pheochromocytoma are widely used as neural model cells. The PC 12 cells were cultured in a D-MEM culture medium containing 5% bovine serum and 5% horse serum. The ceramide-containing matter was dissolved in dimethyl-sulfoxide (DMSO), and the resulting solution was added to the cells in such a way as to give an amount of 30 μg/mL for culture for 5 minutes, 10 minutes, 30 minutes and 60 minutes. After culture, the cells were collected for estimation of the phosphorylation ERK1/2 (phospho ERK1/2) level important for memory formation by means of the western blot method using anti-phospho ERK1/2 antibody and/or anti-total ERK1/2 antibody. The results are shown in FIG. 7. By use of the ceramide-containing matter, there was observed a rise of the phosphorylation level of ERK1/2 with a peak found after the elapse of 10 minutes: the activation of ERK1/2.

Effect of the Ceramide-containing Matter on the MEK-ERK Signaling System in Neural Model Cells (FIG. 8)

[0105] An inhibitor of MEK that was the upstream kinase of extracellular signal-regulated kinase (ERK1/2) was used to make an estimation of the effect of the ceramide-containing matter on the MEK/ERK signaling system in PC-12 cells. The PC-12 cells were cultured in a D-MEM culture medium containing 5% bovine serum and 5% horse serum. The MEK inhibitor UO126 was dissolved in dimethyl-sulfoxide (DMSO), and the resulting solution was added to the cells in such an amount as to give 10 μM, and 30 minutes after that, the ceramide-containing matter dissolved in DMSO was added to the cells in such an amount as to give 30 μg/mL for 10-minute culture. After culture, the cells were collected for estimation of the phosphorylation ERK1/2 (phospho ERK1/2) level important for memory formation by means of the western blot method using anti-phospho ERK1/2 antibody and/or anti-total ERK1/2 antibody. The results are shown in FIG. 8. Note here that a sample treated with nobiletin (30 μM) was used as a positive control. By use of the ceramide-containing matter, the ERK1/2 phosphorylation level was observed to rise, but that rise was kept low by the pretreatment with the MEK inhibitor UO126, indicating that the ceramide-containing matter activates the MEK-ERK signaling system.

[0106] Set out below are the effects of the ceramide-containing matter on the dementia model mice and/or the neural model cells disclosed in Example 3.

(1) The ceramide-containing matter could result in improvements of memory impairments in dementia model mice to which N-methyl-D-aspartate (NMDA) receptor blocker MK-801 was administered.
(2) The ceramide-containing matter could result in a rise in the phosphorylation level of extracellular signal-regulated kinase (ERK1/2) important for memory formation in neural model cells or PC12 cells: the activation of ERK1/2.

[0107] According to the present invention, therefore, it is possible to provide a composition containing ceramide(s) obtained from whole apples and/or apple juice extraction residues as an effective ingredient for improving memory impairments and/or increasing the phosphorylation level of extracellular signal-regulated kinase (ERK1/2). It is here noted that the foregoing may be applied to not only Product 1 of Example 1 but also the general spectrum of ceramides derived from apples.

EXAMPLE 4

[0108] Set out in Example 4 are ursolic acid as Product 2 and/or recovered ethanol and/or a polyphenol solution as Product 3, all obtained in intermediate steps comprising the aforesaid ceramide steps 2, 3 and 4.

Ceramide Step 2 of Extracting Ursolic Acid as Product 2 from the Adsorbent Treatment Liquid (FIGS. 1, 10 and/or 14)

[0109] Product 2 was extracted by means of centrifugation or solid-liquid separation using suction filtration, preferably an analytical filter paper, because precipitates settled down in a solution stemming from the adsorbent treatment liquid obtained from the adsorbent treatment and let standing for one night. The yield of Product 2 is 0.9% to 1.5% as calculated on the basis of 1 kg of the original water treatment/dried residues.

Analysis of Ursolic Acid by Thin-layer Chromatography

[0110] Product 2 extracted in the foregoing ceramide step 2 was dissolved in a solvent comprising a 4:1 chloroform/methanol mixture solvent, and the resulting solution was coated on a silica gel thin layer to carry out thin-layer chromatography using a 65:25:4 chloroform/methanol/water mixture solvent as a developing solvent, after which a copper/phosphoric acid coloration reagent was sprayed on the surface of the silica gel thin layer and then heated for coloration. As a result, it has been found that Product 2 precipitated and extracted in ceramide step 2 is ursolic acid.

Ceramide Step 3: Recovery of Ethanol by the Concentration under Reduced Pressures of the Precipitate-free Liquid Obtained in Ceramide Step 2 (FIGS. 1, 11 and/or 14)

[0111] The precipitate-free liquid obtained in ceramide step 2 can be concentrated under reduced pressures to obtain a concentrate and recover ethanol that may be reused. The recovered ethanol may be reused as ethanol in ceramide step 1. In a typical example, the ethanol recovery rate was 70%. As a result of measuring the water content of recovered ethanol by means of a density indicator, it has been found to keep the original water content intact, meaning that the ethanol may be reused with no need for any additional step.

Ceramide Step 4: Preparation of a Polyphenol Solution of Product 3 by Water Addition and Heat Insulation of the Concentrate (FIGS. 1, 12 and/or 14)

[0112] In ceramide step 4, water is added to the concentrate obtained in ceramide step 3 followed by thermal insulation for solid-liquid separation of a precipitating solid ingredient from an aqueous solution. The liquid portion is polyphenol-containing matter. In a typical example, 0.5 liter of the concentrate was added with water in a 4-fold amount or 2 liters, and the obtained precipitate was washed with 2 liters of fresh water, whereupon the filtrate was obtained as a polyphenol solution in a combined amount of 4.5 liters.

Analysis of the Polyphenol Solution Obtained in Ceramide Step 4

[0113] In ceramide step 4, a polyphenol solution is obtained as a solution portion in which the ceramide-containing matter precipitating after water addition and heat insulation is subjected to solid-liquid separation. As a result of measuring the content of polyphenol contained in this solution by the Folin-Denis method, it has been found that the amount of polyphenol contained in 100 mL solution is 150 mg as calculated as the amount of catechin. Therefore, the polyphenol solution had a concentration of 0.15%, and was obtained in an amount of 6.75 grams from 1 Kg of the original water/drying treatment residues.

[0114] As set forth in Example 4, the results obtained in the respective steps from ceramide step 2 to ceramide step 4 are summarized below.

(1) In ceramide step 2, ursolic acid was extracted as Product 2 from the adsorbent-free liquid in an amount of 0.9% to 1.5% per 1 Kg of the original water-treated/dried matter.
(2) In ceramide step 3, the concentrate could be prepared with ethanol recovery in a recovery rate of 70%.
(3) In ceramide step 4, the polyphenol solution was obtained in a solution from which a solid ingredient precipitating by water addition and heat insulation of the concentrate in a volume of 4.5 L per 1 Kg of the residues with a polyphenol concentration of 150 mg/100 mL as calculated on a catechin basis.

EXAMPLE 5

[0115] In Example 5, the ceramide-containing matter extracted in the foregoing ceramide step 4 was subjected to separation/purification and instrumental analysis.

Separation/Purification of the Ceramide-containing Matter by Silica Gel Column Chromatography (FIGS. 13 and/or 14)

[0116] Zero point two (0.2) gram of the ceramide-containing matter extracted in the foregoing ceramide step 4 was taken and dissolved in 2 mL of a solvent in which chloroform and methanol were mixed in 4:1. Then, silica gel suspended in a 4:1 chloroform/methanol mixture solvent, preferably 230 to 400 mesh silica gel available from Merck & Co. was filled up in a glass column tube having a diameter of 30 mm in such a way as to provide a height of 420 mm. Note here that the column volume was about 300 mL. A solution in which the foregoing ceramide matter was dissolved was adsorbed onto the tip of the silica gel column, and a 4:1 chloroform/methanol mixture solvent was allowed to flow down the column in a flow rate of 2 mL/min. Then, a solution flowing out of the lower end of the column was subdivided in test tube Nos. 1 to 40, each in an amount of 10 mL. Of these test tubes, test tube Nos. 15 to 34 were each spotted on a silica gel thin-layer plate (silica gel 60F254 of 10 cm×20 cm available from Merck & Co., and then developed using a developing solvent comprising a 65:25:4 chloroform/methanol/water mixture solvent. After development, an anthrone sulfate reagent was sprayed and heated for coloration. The thin-layer plate was spotted at both its ends with a commercially available ceramide standard (derived from soy beans as a standard substance for comparison of mobility and color tone. Ceramide was separated from test tube No. 31 to No. 33 by column chromatography of the ceramide-containing matter.

Separation and Purification of Ceramide by Preparative High Speed Liquid Chromatography (FIGS. 13 and/or 14)

[0117] The content of Test tube No. 33 collected from test tube No. 31 subjected to the foregoing separation by silica gel column chromatography was concentrated under reduced pressure, and dissolved in a 95:5 methanol/water mixture solvent. The preparative high speed chromatography was carried out by connecting a high speed liquid chromatography system with a preparative reversed chromatography system, preferably TSKGELODS-80TS (available from TOSOH CORPORATION) having a column size of 8 mm in diameter and 300 mm in length, and using as an eluent a 95:5 methanol/water mixture solvent at a flow rate of 2 mL/min. for detection at 210 nm. An ingredient flowing out at a holding time of 19.96 minutes was collected and concentrated to dryness for instrumental analysis.

Analysis of Ceramide by Infrared Adsorption Spectra

[0118] Measured here were the infrared adsorption spectra of the ingredient flowing out at the holding time of 19.96 minutes by the foregoing preparative high speed liquid chromatography, and for the infrared adsorption spectra, a sample was regulated according to the KBr-Disc method, and a Fourier transform infrared analyzer was then used to measure transmittances (T%) from 4000 cm-1 to 400 cm-1. The spectra conformed to those of the ceramide.

Analysis of Ceramide by Nuclear Magnetic Resonance Spectra

[0119] An ingredient flowing out at the holding time of 19.96 minutes in the foregoing preparative high speed liquid chromatography was dissolved in a solution comprising a 4:1 heavy chloroform/heavy methanol mixture, and nuclear magnetic resonance spectra were measured using a 270 MHz Fourier transform nuclear resonance device (available from JEOL) with tetramethylsilane as the internal standard.

[0120] Set out below are the results of separation/purification or instrumental analysis of ceramide contained in the ceramide-containing matter disclosed in Example 5.

(1) The ceramide was separated from the ceramide-containing matter and purified by silica gel column chromatography followed by preparative high speed liquid chromatography.
(2) The structure of the ceramide separated and purified by the foregoing chromatography means are analyzed by infrared adsorption spectra and/or nuclear magnetic resonance spectra. These spectra conformed to those of the ceramide.

APPLICABILITY TO THE INDUSTRY

[0121] The present invention provides an extraction method/process in which matter or material obtained by water treatment and drying of whole apples and/or apple juice extraction residues is used as the raw or starting material, and then treated with ethanol thereby extracting ceramide-containing matter or ceramide and/or ursolic acid and/or a poly-phenol solution contained in the resulting ethanol treatment liquid. In this process, ingredients contained in the ethanol treatment liquid can be utilized, and the ethanol used for extraction can be recovered and reused. By the present invention it is possible to use whole apples thrown away without being eaten raw or processed as a raw material, resulting in industrial use of apple juice resides that are discarded as unavailable resource or apply loads to the environment.

[0122] In the present invention, it has been verified on substance and gene expression levels that the ceramide-containing matter extracted at the foregoing respective method/process steps increases or enhances production of hyaluronic acid by the human skin culture cells and production of type II collagen by the human cartilage cells. Human-derived hyaluronic acid or type II collagen is a substance that has an important role in each human tissue and a direct effect on maintenance of health and appearance or improvements in the quality of life. It has been found for the first time that the ingredients contained in whole apples and/or apple juice resides have an effect on enhancement of production of hyauronic acid or type II collagen. Whole apples and/or apple juice extraction residues are available in abundance, and the useful ingredients obtained in the process steps of FIGS. 1 through 14 can be industrially utilized as foodstuff material or cosmetics material.

[0123] In the present invention, the ceramide-containing matters extracted in the respective process steps brought about an improvement in memory impairments in dementia model mice and a rise of the level of phosphorylation of extracellular signal-regulated kinase (ERK1/2) important for memory formation in rat-derived neural model cells: activation of ERK1/2. Improvements of dementias or memory impairments have a direct influence on maintenance of health or an improvement in the quality of life with much more patients in the aging society in mind. It has been found for the first time that the ingredients contained in whole apples and/or apple juice extraction residues have an effect on improvements of dementias and memory impairments. Whole apples and/or apple juice extraction residues are available in abundance; useful ingredients obtained in the respective process steps of FIGS. 1 to 14 can be used in an industrial scale as materials for health foodstuff and pharmaceuticals.