WO2023063424A1

WO2023063424A1 - Ceramide-containing composition and method for producing same

Info

Publication number: WO2023063424A1
Application number: PCT/JP2022/038436
Authority: WO
Inventors: 有宏金; 裕晃山田; アンガサンジャヤ，; 英一金; 敦史山津; 武祚金
Original assignee: 株式会社ファーマフーズ
Priority date: 2021-10-15
Filing date: 2022-10-14
Publication date: 2023-04-20

Abstract

Provided is a composition containing a ceramide derived from a natural substance.　The present invention provides a composition containing a polyphenol and a ceramide. The invention of the present application further provides a composition containing a ceramide derived from wine fermentation lees. Also provided is a method for producing a ceramide-containing composition, the method comprising a step for washing wine fermentation lees and a step for extracting the resulting washing residue using a polar solvent.

Description

Ceramide-containing composition and method for producing the same

The present invention relates to compositions containing ceramide. More specifically, it relates to compositions containing polyphenols and ceramides, especially free ceramides, and methods of making the same.

　Ceramide is a type of sphingolipid and a major component of stratum corneum intercellular lipids. Ceramide is expected to have effects such as skin barrier effect (surface protection), moisturizing effect, moisture regulation, elasticity retention, collagen protection, and antioxidant effect when applied to the skin or taken orally. Many cosmetic products and functional foods are provided.

Among ceramides, plant-derived ceramide raw materials have been put into practical use from many plants such as konnyaku and rice, and all of them contain glucosylceramide (sugar ceramide) as the main component and are mainly used as raw materials for food.

On the other hand, these glucosylceramides are non-human ceramides and differ in structure from human ceramides. In addition, human ceramide is in a free form (free ceramide) in which sugars and phosphoric acids are not bound, and is not contained in normal living cells, so it is a component that hardly exists in the natural world. Therefore, the development of free ceramides derived from natural products is desired.

The present inventors have found a novel composition useful for health and/or beauty, containing polyphenols and ceramide (especially free ceramide). The present inventors also found that a composition containing ceramide (particularly, free ceramide) in addition to polyphenols can be obtained by subjecting fermented lees produced during wine brewing to a specific treatment. rice field. Therefore, the present invention provides a composition derived from a natural product containing such polyphenols and ceramides, and a method for producing the same.

Therefore, according to main aspects of the present invention, the following inventions are provided.
(Item 1)
A cosmetic and/or oral composition containing polyphenols and ceramides.
(Item 2)
The composition according to the preceding items, wherein the ceramide comprises free ceramide and glucosylceramide.
(Item 3)
A composition according to any one of the preceding items, wherein the composition comprises at least about 1% by weight of free ceramide.
(Item 4)
A composition according to any one of the preceding items, wherein said composition comprises at least about 1% by weight of glucosylceramide.
(Item 5)
A composition according to any one of the preceding items, wherein the composition comprises at least about 1% by weight of polyphenols.
(Item 6)
Any one of the preceding items, wherein the weight ratio of free ceramide, glucosylceramide, and polyphenol in the composition is about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. 13. The composition of claim 1.
(Item 7)
A composition according to any one of the preceding items, wherein the weight ratio of free ceramide, glucosylceramide and polyphenol in the composition is about 1.0: about 1.0: about 1.0. (Item 8)
A composition according to any one of the preceding items, wherein the composition has a pH of less than about 5.
(Item 9)
The composition according to any one of the preceding items, comprising at least about 10 mg resveratrol per about 100 g.
(Item 10)
The composition according to any one of the above items, which is for food.
(Item 11)
The composition according to any one of the above items, which is used for cosmetics.
(Item 12)
The composition according to any one of the above items, wherein the polyphenol and the ceramide are derived from wine fermentation lees.
(Item 13)
A composition according to any one of the preceding items for promoting keratinocyte differentiation.
(Item 14)
A composition according to any one of the preceding items for promoting ceramide synthesis.
(Item A1)
A ceramide-containing composition derived from wine fermentation lees.
(Item A2)
A composition according to any one of the preceding items, wherein the ceramide-containing composition comprises at least about 1% by weight of free ceramide.
(Item A3)
A composition according to any one of the preceding items, wherein the ceramide-containing composition comprises at least about 1% by weight of glucosylceramide.
(Item A4)
A composition according to any one of the preceding items, wherein the ceramide-containing composition comprises at least about 1% by weight of polyphenols.
(Item A5)
Any one of the preceding items, wherein the weight ratio of free ceramide, glucosylceramide, and polyphenol in the composition is about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. 13. The composition of claim 1.
(Item A6)
The composition according to any one of the preceding items, wherein the weight ratio of free ceramide, glucosylceramide and polyphenol in the ceramide-containing composition is about 1.0: about 1.0: about 1.0.
(Item A7)
A composition according to any one of the preceding items, wherein the ceramide-containing composition has a pH of less than about 5.
(Item A8)
The composition according to any one of the preceding items, comprising at least about 10 mg resveratrol per about 100 g.
(Item A9)
The composition according to any one of the above items, which is for food.
(Item A10)
The composition according to any one of the above items, which is used for cosmetics.
(Item A11)
A composition according to any one of the preceding items for promoting keratinocyte differentiation.
(Item A12)
A composition according to any one of the preceding items for promoting ceramide synthesis.
(Item B1)
A method for producing a ceramide-containing composition, comprising:
A step of washing the wine fermented lees;
and extracting the residue obtained by said washing with a polar solvent.
(Item B2)
The method according to the preceding items, wherein the polar solvent comprises ethanol.
(Item B3)
A method according to any one of the preceding items, wherein the ethanol concentration is about 95%.
(Item B4)
A method according to any one of the preceding items, wherein the washing step is performed with water.
(Item B5)
A method according to any one of the preceding items, further comprising the step of pH-adjusting the extract obtained by the step of extracting.
(Item B6)
A method according to any one of the preceding items, wherein said pH adjustment is performed with tartaric acid or citric acid.
(Item C1)
A method for promoting keratinocyte differentiation, comprising administering a composition according to any one of the preceding items.
(Item C2)
A method for promoting ceramide synthesis, comprising administering a composition according to any one of the preceding items.

In the present disclosure, it is intended that one or more of the above features may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description, if necessary.

It should be noted that features and remarkable actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following embodiments of the invention and the drawings.

According to the method of the present invention, a composition containing polyphenols and ceramides, especially human-type free ceramides, can be obtained. In particular, when such a composition is obtained from fermented wine lees, it uses fermented wine lees, which is a highly safe natural material. It has excellent safety and high utility value without adversely affecting the environment.

FIG. 1 is a graph according to an embodiment showing the difference between the composition of ceramide molecular species in wine starch and the composition of ceramide molecular species in soybean. FIG. 2 is a graph according to one embodiment showing the difference between the amount of ceramide obtained from wine lees extract and the amount of ceramide obtained from wine. FIG. 3 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine the washing step in the process for producing a ceramide-containing composition. The left side of FIG. 3 shows the case where the ceramide extract was extracted from the starting wine lees with 95% ethanol, and the right side of FIG. 3 shows the case where the starting wine lees were washed with 50% ethanol. FIG. 4 is a graph according to one embodiment showing differences in composition of ceramide-containing compositions due to differences in washing processes. FIG. 4 shows the content (mg) of each component. FIG. 5 shows the difference in the composition of the ceramide-containing composition due to the difference in the washing process (when the ceramide extract is extracted from the raw wine lees with 95% ethanol and when the raw wine lees are washed with 50% ethanol). 4 is a graph according to one embodiment; FIG. 5 shows the ratio of each component. FIG. 6 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine the washing step in the process for producing a ceramide-containing composition. It shows the case where the raw wine lees are washed with water, 10%, 30%, or 50% ethanol. FIG. 7 is a graph according to an embodiment showing the difference in the composition of the ceramide-containing composition due to the difference in the washing process (when the raw wine starch was washed with water, 10%, 30%, or 50% ethanol). . The left side of FIG. 7 shows the ratio of each component, and the right side of FIG. 7 shows the content (mg) of each component. FIG. 8 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine the washing step in the process for producing a ceramide-containing composition. It shows the case where the raw wine lees are washed with water at 40°C or 60°C. FIG. 9 is a graph according to one embodiment showing the difference in the composition of the ceramide-containing composition due to the difference in the washing process (when the raw wine starch is washed with water at 40° C. or 60° C.). The left side of FIG. 9 shows the ratio of each component, and the right side of FIG. 9 shows the content (mg) of each component. FIG. 10 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine the washing step in the process for producing a ceramide-containing composition. Raw wine lees are shown washed with water for 30, 60, or 180 minutes. FIG. 11 is a graph according to one embodiment showing the difference in the composition of the ceramide-containing composition due to the difference in the washing process (when the raw wine lees are washed with water for 30, 60, or 180 minutes). The left side of FIG. 11 shows the ratio of each component, and the right side of FIG. 11 shows the content (mg) of each component. FIG. 12 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine the washing step in the process for producing a ceramide-containing composition. It shows the case where the raw material wine sediment was washed with 10 times, 8.3 times, and 6.7 times the amount of water. FIG. 13 shows the difference in the composition of the ceramide-containing composition due to the difference in the washing process (when the raw wine starch was washed with 10 times, 8.3 times, and 6.7 times the amount of water) according to one embodiment. graph. The left side of FIG. 11 shows the ratio of each component, and the right side of FIG. 11 shows the content (mg) of each component. FIG. 14 is a schematic diagram showing a process flow for producing a ceramide-containing composition, which was used to examine pH adjustment in the process for producing a ceramide-containing composition. It shows the case where tartaric acid, citric acid, or sodium carbonate is added to the ceramide extract extracted from the raw wine lees. FIG. 15 is a photograph according to one embodiment showing the difference in color of the ceramide-containing composition due to the difference in pH. FIG. 16 is a graph according to one embodiment showing the effect of a ceramide-containing composition on human epidermal cell proliferation. When human epidermal cells (Hacat cells) were cultured in a medium supplemented with various concentrations of the ceramide-containing composition, cell growth activity was significantly increased in the group treated with the ceramide-containing composition compared to the non-supplemented medium. FIG. 17 is a graph according to one embodiment showing the amount of resveratrol contained in red wine, wine lees (wine crystals), and ceramide-containing composition (wine ceramide). FIG. 18 is a graph showing mRNA expression levels of differentiation markers (Pro-Filaggrin and Loricrin) when wine ceramide according to one embodiment of the present invention was added to a human epidermal keratinocyte Hacat cell line and cultured for 72 hours. FIG. 19 is a graph showing the mRNA expression level of ceramide synthesis pathway marker (SPT: Serine-Palmitoyl Transferase) when wine ceramide according to one embodiment of the present invention was added to human epidermal keratinocyte Hacat cell line and cultured for 72 hours. is. FIG. 20 shows a differentiation marker (Pro-Filaggrin) and ceramide synthesis pathway when wine ceramide, glucosylceramide, and free ceramide according to one embodiment of the present invention were added to human epidermal keratinocyte Hacat cell line and cultured for 72 hours. It is a graph which shows the mRNA expression level of a marker (SPT). FIG. 21 shows that wine ceramide according to one embodiment of the present invention and ceramides obtained from raw materials other than wine (rice ceramide, pine ceramide, and konjac ceramide) were added to human epidermal keratinocyte Hacat cell line, and 72 hours. 1 is a graph showing mRNA expression levels of a differentiation marker (Pro-Filaggrin) and a ceramide synthesis pathway marker (SPT) when cultured. FIG. 22 is a photograph of a cell section when wine ceramide according to one embodiment of the present invention was added to 3D-cultured keratinocytes and cultured for 48 hours. The amount of ceramide in the cell section was stained with hematoxylin and eosin and observed under a phase-contrast microscope. The graph shows mean ± SD values of skin thickness in μm (N=4). Wine ceramide according to one embodiment of the present invention significantly increased epidermal thickness. FIG. 23 is a photograph of a cell section when wine ceramide according to one embodiment of the present invention was added to 3D-cultured keratinocytes and cultured for 48 hours. The amount of ceramide in the cell section was immunostained with an anti-ceramide antibody and observed under a phase-contrast microscope. The graph shows the mean±SD value of relative fluorescence density in 3D cultured keratinocytes (N=4). Wine ceramide according to one embodiment of the present invention significantly increased the amount of ceramide in the skin.

The present disclosure will be described below while showing the best mode. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Thus, articles in the singular (eg, “a,” “an,” “the,” etc. in the English language) should be understood to include their plural forms as well, unless otherwise stated. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

Definitions of terms and/or basic technical contents particularly used in this specification are explained below as appropriate.

As used herein, "about" means ±10% of the following numerical value.

As used herein, “ceramide” is a kind of sphingolipid and refers to a group of compounds in which sphingosine and fatty acid are amide bonded. As used herein, "ceramide" also includes ceramide derivatives. Ceramide is a compound having a structure (--NH--CO--) in which an amino group (--NH ₂ ) of a sphingoid base is bonded to a carboxyl group (--COOH) of a fatty acid. The alcoholic hydroxyl group (--OH) of the sphingoid base of ceramide is further combined with a polar group such as sugar and phosphoric acid to form glycosphingolipids and phosphosphingolipids, respectively. Here, those bound with sugars are particularly called glycosylceramides, and particularly when the sugar is glucose, they are called glucosylceramides. Ceramide is called free ceramide when sugar, phosphoric acid, etc. are not bound to ceramide. Since free ceramide is generally an intermediate metabolite in the sphingolipid synthesis system, it exists only in minute amounts, especially in animals and plants. Galactosylceramide, glucosylceramide, and sphingomyelin, in which ceramide is bound with sugar or phosphoric acid, are extracted from animals and plants, but because the sugar or phosphoric acid is bound, they are not found in intercellular lipids. orientation is different from that of free ceramide.

As used herein, "polyphenol" is a general term for compounds having multiple phenolic hydroxyl groups in the molecule, specifically flavonols, flavones, isoflavones, tannins, catechins, quercetin, anthocyanins, anthocyanidins, leucoanthocyanidins. , proanthocyanidins, propyl gallate, catechin, rutin, quercetin, ferulic acid, resveratrol, procyanidins, theaflavin, curcumin, pycnogenol, rosmarinic acid, ellagic acid, quinic acid, chlorogenic acid, hydroxytyrosol and the like.

As used herein, "washing" refers to washing away and removing impurities by stirring and mixing wine fermented lees (wine lees) and liquid.

As used herein, "extraction" means separating the analyte contained in the solution from other impurities, and is also referred to as solvent extraction or liquid-liquid extraction.

(preferred embodiment)
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. Also, the following embodiments of the disclosure may be used singly or in combination.

In one aspect of the present invention, a cosmetic and/or oral composition containing polyphenols and ceramides is provided. In another aspect of the present disclosure, a ceramide-containing composition derived from wine fermentation lees is provided. Since both the cosmetic and/or oral intake composition containing polyphenol and ceramide and the ceramide-containing composition derived from fermented wine lees contain ceramide, they are not particularly mentioned in the present specification. Except for this, it can be collectively referred to as a "ceramide-containing composition". Moreover, in this specification, the ceramide-containing composition of the present invention may be referred to as "wine ceramide".

Ceramide is a type of sphingolipid and includes a group of compounds in which sphingosine and fatty acid are amide bonded. In addition, ceramide is a non-permeable compound having one or more long-chain straight and/or branched alkyl or alkenyl groups, at least two hydroxyl groups, and one or more amide groups (and/or amino groups) in the molecule. It may be an ionic amphiphilic substance. Ceramide may also be a substance in which a phosphatidylcholine residue or a sugar residue is bound to the hydroxyl group of the nonionic amphiphilic substance (a type of so-called ceramide derivative).

In one embodiment, ceramides include free ceramides and glucosylceramides. For example, in a preferred embodiment, ceramides include plant-derived ceramides, preferably wine fermentation lees-derived ceramides.

The free ceramide is not particularly limited as long as it can be used for cosmetics, external skin preparations, or food. 6I, and natural free ceramides such as ceramide 6II. The ceramide-containing composition of the present invention can be obtained using materials that do not adversely affect the human body, so safety is important for food and skin applications. It is effective as a raw material for the intended use. In addition, since it contains human-type free ceramide, it is also suitable as a raw material for foods such as health foods. In one embodiment of the present disclosure, the ceramide-containing composition of the present invention can contain only one type of these free ceramides or a combination of two or more types in any mixing ratio.

The ceramide in the ceramide-containing composition of the present invention has a barrier function that prevents moisture from evaporating from the skin, a function that restores the barrier function, a function that imparts firmness and elasticity to the hair, a function that promotes exfoliation of keratin, a function that smoothes the skin, and a skin The ceramide-containing composition of the present invention has various functions such as a function of exerting a moisturizing effect when applied to hair and a function of exerting a ceramide production promoting effect, and the function of the ceramide-containing composition of the present invention is one of these functions or It is not limited to two or more, and may have a function as a ceramide other than these functions.

In one embodiment of the present invention, the ceramide-containing composition of the present invention may be applied to the skin or used as food. In either case, it is possible to enjoy the above-mentioned functions including moisturizing of the skin, improvement of dark spots and wrinkles, protection against ultraviolet rays, and the like.

In one embodiment of the present invention, the ceramide-containing composition of the present invention promotes differentiation of keratinocytes in the skin and/or promotes de novo synthesis of ceramide by being applied to the skin and/or ingested as food. can be promoted. The ceramide-containing composition of the present invention can, for example, increase gene expression of keratinocyte differentiation markers. The keratinocyte differentiation marker is not particularly limited as long as it can confirm keratinocyte differentiation, but preferably includes a genetic marker. factor (NMF)) and Loricrin (a protein that serves as a framework for normal formation of the stratum corneum).

In addition, the ceramide-containing composition of the present invention can promote ceramide synthesis, for example, by enhancing the expression of genes associated with ceramide synthesis in the ceramide synthesis pathway. De novo synthesis of ceramide is based on Serine + Palmitoyl CoA
→ 3-keto-sphinganine → Sphinganine → Dihydroceramide → Ceramide is known. The differentiation marker of the ceramide synthesis pathway is not particularly limited as long as it is involved in the ceramide synthesis pathway, but preferably includes a genetic marker. (enzyme). SPT catalyzes the conversion of Serine+Palmitoyl CoA to 3-keto-sphinganine in the ceramide synthesis pathway.

In addition, the ceramide-containing composition of the present invention can increase skin thickness and promote skin turnover by promoting the increase and differentiation of keratinocytes.

In one embodiment of the present invention, the ceramide-containing composition of the present invention comprises at least about 0.01 wt% or more free ceramide, preferably at least about 0.1 wt% or more, at least about 0.3 wt% or more. , at least about 0.5% by weight or more, or at least about 0.8% by weight or more of free ceramide, and more preferably at least about 1% by weight or more of free ceramide.

In one embodiment of the present invention, the ceramide-containing composition of the present invention comprises at least about 0.01 wt% or more of glucosylceramide, preferably at least about 0.1 wt% or more, at least about 0.3 wt% or more. , at least about 0.5% by weight or more, or at least about 0.8% by weight or more of glucosylceramide, and more preferably at least about 1% by weight or more of glucosylceramide.

Polyphenol is a compound having a plurality of phenolic hydroxyl groups in the molecule, and the type of polyphenol contained in the composition of the present invention is not particularly limited. Examples include flavonol, flavone, isoflavone, tannin, catechin, quercetin, Anthocyanin, anthocyanidin, leucoanthocyanidin, proanthocyanidin, propyl gallate, catechin, rutin, quercetin, ferulic acid, resveratrol, procyanidin, theaflavin, curcumin, pycnogenol, rosmarinic acid, ellagic acid, quinic acid, chlorogenic acid, hydroxytyrosol etc. In the composition of the present invention, these polyphenols may be contained singly or in combination of two or more.

In one embodiment, the polyphenol content in the compositions of the present invention is at least about 0.01 wt% or more, preferably at least about 0.1 wt% or more, at least about 0.3 wt% or more, at least about It can be at least 0.5% by weight, or at least about 0.8% by weight or more, and more preferably at least about 1% by weight or more.

In one embodiment of the present invention, the composition of the present invention may comprise resveratrol as a polyphenol in an amount of at least about 1 mg, at least about 3 mg, at least about 5 mg, at least about 7 mg per about 100 g of the composition of the present invention. , or at least about 10 mg resveratrol.

In one embodiment of the present invention, the composition of the present invention contains glucosylceramide, free ceramide, and polyphenol as described above in a weight ratio of glucosylceramide:free ceramide:polyphenol=about 1.0:about 0.5. 5 to about 3.0: about 1.0 to about 15.0, preferably about 1.0: about 0.7 to about 2.4: about 1.4 to about 11.0, more preferably about 1 .0: about 1.0: about 1.0. Regarding ceramide as a food raw material, many plant-derived ceramides such as konjac and rice are produced, but since all of them contain glucosylceramide as the main component, the ceramide-containing composition of the present invention has the same level of glucosylceramide. It is useful in that it contains less or more free ceramide.

In another embodiment, the compositions of the present invention contain ceramides and polyphenols in a weight ratio of total ceramides: polyphenols of about 1.0: about 0.5 to about 5.0, preferably about 1.0: It can be from about 0.7 to about 4.0.

In another aspect of the present invention, a method for producing a ceramide-containing composition, comprising the steps of washing wine fermented lees and extracting the residue obtained by the washing with a polar solvent. provided.

The method for producing a ceramide-containing composition of the present invention comprises washing wine fermentation lees (bitartrate) obtained during wine brewing, and then extracting the residue with a polar solvent to obtain a fermented product extract. Characterized by There are no particular restrictions on the grape variety, the type of yeast, or the wine production method used as the raw material for the wine fermented lees. It can be used as wine fermentation lees. Wine yeast used for wine fermentation is not particularly limited, but natural yeast and cultured yeast can be used. Therefore, no matter what type of wine lees is used as a starting material, a composition with an increased content of ceramide or glucosylceramide can be produced by the method for producing a ceramide-containing composition of the present invention.

In one embodiment, polar solvents such as water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, and ethyl acetate can be used as polar solvents. Two or more of these solvents may be mixed. Preferably, the ceramide-containing composition of the present invention can be efficiently extracted by using ethanol or hydrous ethanol, which is a mixture of ethanol and water, as the polar solvent.

When hydrous ethanol is used as the polar solvent, the concentration of ethanol is not particularly limited, for example, the ethanol concentration can be about 10% to about 99% (wt/wt), preferably the ethanol concentration is at least about 70%. % or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 93% or more, about 95% or more, about 98% or more, about 99% or more (wt/wt) can be done.

In one embodiment, the extraction temperature in the extraction step is about 30°C to about 80°C, preferably about 40°C to about 60°C. The extraction time can be about 1 hour to about 8 hours, preferably about 3 hours to about 6 hours.

Any method such as stirring extraction, continuous extraction, immersion extraction, countercurrent extraction, or supercritical extraction can be used as the extraction method, and any device can be used at room temperature or under reflux heating. Preferably, in the method of the present invention, stirring extraction can be performed using an oar-type or propeller-type stirrer as the extraction method. In one embodiment, when stirring extraction is performed, wine lees and a solvent (such as ethanol) can be stirred and extracted in an extraction tank while being heated to a predetermined temperature. After stirring for a predetermined period of time, the mixture is filtered, and the filtrate can be used as a ceramide fraction (the ceramide-containing composition of the present invention). The scale of extraction can be set appropriately, so in another embodiment, a beaker is placed in a water bath, and wine lees and a solvent (such as ethanol) are stirred in the beaker for extraction. .

In one embodiment, the wine fermentation lees (wine lees) can be washed with water in the washing step. In one embodiment, in the washing step, washing can be performed while stirring the fermented wine lees and water using an oar-type or propeller-type agitator. In one embodiment, the fermented wine lees and water can be stirred while being heated. After stirring for a period of time, it can be filtered and the residue used in the extraction process. Examples of water include purified water, sterilized water, and tap water. Purified water or sterilized water is preferable because it contains few impurities. In this case, the washing temperature can be about 30°C to about 80°C, preferably about 40°C to about 60°C. The cleaning time can be about 5 minutes to about 6 hours, preferably about 1 hour to about 2 hours.

In one embodiment of the present invention, the method for producing the ceramide-containing composition of the present invention can further adjust the pH of the extract obtained by the extraction step with a polar solvent. By such pH adjustment, the color of the obtained ceramide-containing composition can be adjusted, and preferably a desired wine color can be obtained. Such pH adjustment can be performed with an acid or alkali, and the type of acid or alkali is selected within a range that does not impair the effects of the ceramide-containing composition of the present invention. , food for specified health use, supplements, pharmaceuticals, quasi-drugs, or external skin preparations, but not limited to, for example, phosphoric acid, tartaric acid, citric acid, or sodium carbonate can be done.

In one embodiment, the ceramide-containing compositions of the present invention can be pH-adjusted for color depending on the desired application, wherein the pH-adjustment as described above reduces the pH of the ceramide-containing composition to, for example, less than about 5. , less than about 4.8, less than about 4.0, or less than about 3.5.

In addition, for the extraction, separation, purification, identification, etc. of the ceramide-containing composition of the present invention, a wide range of normal operations can be applied. It is also possible to adjust the pH, mix with desired excipients as necessary, and perform concentration, heat sterilization, freeze-drying, and the like.

In one preferred embodiment of the present invention, the ceramide-containing composition of the present invention is obtained by washing wine fermented lees (wine lees) with preferably about 10 times the amount of water at about 60°C for about 1 hour, and then removing the residue. Preferably, it can be obtained by performing extraction with an ethanol solution of about 95% at about 40° C. for about 6 hours. Further, in one preferred embodiment of the present invention, the ceramide-containing composition of the present invention can adjust the pH of the extract obtained as described above to less than about 4.0. Furthermore, in one preferred embodiment of the present invention, the ceramide-containing composition of the present invention is mixed with an excipient such as cyclodextrin, further concentrated by replacing ethanol with water, heat sterilized at about 120 ° C., It can be lyophilized and powdered.

In one preferred embodiment of the present invention, the ceramide-containing composition of the present invention is obtained by the production method described above, and contains glucosylceramide, free ceramide, and polyphenol at a weight ratio of about 1.0:1. .0: about 1.0 ratio. Moreover, in one preferred embodiment of the present invention, the ceramide-containing composition of the present invention can contain at least about 10 mg of resveratrol per about 100 g of the composition of the present invention by being obtained by the method described above. .

The ceramide-containing composition produced by the method of the present invention can be applied or blended for various uses. Applications include, for example, cosmetics, pharmaceuticals, reagents, and compositions for oral intake (eg, health foods, foods with function claims, foods for specified health uses, supplements, and foods for specific uses).

In one embodiment, the product containing the ceramide-containing composition of the present invention may contain excipients, carriers, additives, etc., the types of which are commonly used and pharmaceutically acceptable It is not particularly limited as long as it is a substance, and the blending amount thereof can be changed as appropriate. Examples of excipients include sodium chloride, sodium citrate, etc. Examples of carriers include sterilized water, physiological saline, and various buffer solutions. Preservatives, preservatives and the like can be mentioned.

In one embodiment, the dosage form of the pharmaceutical containing the ceramide-containing composition of the present invention is not particularly limited, and may be appropriately selected as necessary. Oral agents such as granules and powders, and parenteral agents such as injections, suppositories, and ointments can be used. The amount of the ceramide-containing composition of the present invention in these pharmaceuticals is not particularly limited, and can be appropriately set according to the type and application of the pharmaceutical.

In one embodiment, compositions for oral intake containing the ceramide-containing composition of the present invention (e.g., health foods, foods with function claims, foods for specified health uses, supplements, and foods for specified dietary uses) optionally contain dextrin. , lactose, starch, and other excipients, flavors, pigments, gelatin, etc., and the ceramide-containing composition of the present invention can be blended. The amount of the ceramide-containing composition of the present invention blended in these compositions for oral intake is not particularly limited, and can be appropriately set according to the type and use of the composition for oral intake. In one embodiment, these compositions for oral ingestion can contain various ingredients depending on the type. In one embodiment, the ceramide-containing composition of the present invention can also be added to foods and drinks that are normally consumed.

In one embodiment, the ceramide-containing composition of the present invention can also be incorporated into external skin preparations including cosmetics, pharmaceuticals, and quasi-drugs. For example, the ceramide-containing composition of the present invention can be used for skin lotions, milky lotions, creams, lotions, soaps, facial cleansers, bath agents, eau de colognes, cosmetic oils, suntan/sunscreen lotions, powders, foundations, perfumes, packs, eye creams, etc. It can be incorporated into creams, eye shadows, mascaras, eyeliners, lipsticks, lip balms, shampoos, rinses, hair dyes, ointments, creams, liquids for external use, and the like.

In one embodiment of the present invention, external skin preparations (including cosmetics, pharmaceuticals, and quasi-drugs) containing the ceramide-containing composition of the present invention contain: Various ingredients used in usual external preparations for skin, e.g., surfactants, fatliquors, hair protectants, moisturizers, polymers, UV absorbers, sequestering agents, solvents, pH adjusters, vitamins, oxidation Antioxidants, antioxidant aids, preservatives, fragrances, oily components, higher alcohols, fatty acids, polar lipids, antibacterial components, viscosity modifiers, pigments, and the like can be blended.

In one embodiment of the present invention, the ceramide-containing composition of the present invention is spray-dried, freeze-dried, or vacuum-dried, and then made into powder, granules, or a solution for use in external skin preparations, food and drink, pharmaceuticals, and the like. can be easily included.

In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "or". When we say "within a range" of "two values" herein, the range includes the two values themselves.
All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

In the above, the present disclosure has been described by showing preferred embodiments for easy understanding. While the present disclosure will now be described based on the examples, the foregoing description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but only by the claims.

(Example 1: Ceramide molecular species in wine sediment)
50 g of wine lees (Bitartrate) was washed with water at 60° C. for 1 hour to obtain a residue. The residue was extracted with 95% ethanol at 40° C. for 6 hours. The composition of ceramide molecular species in this extract was compared with ceramide molecular species similarly obtained from soybean. The results are shown in FIG. As can be seen from these results, ceramide obtained from soybean is only glucosylceramide and does not contain free ceramide, while ceramide obtained from wine starch contains both glucosylceramide and free ceramide. As soybean ceramide, pure soybean-derived glucosylceramide manufactured by Nagara Science Co., Ltd. was used.

Fig. 2 shows the results of confirming the ceramide molecular species and their amounts contained in the wine lees extract and the wine itself. From this result, it can be seen that wine itself contains neither free ceramide nor glucosylceramide, but the extract obtained from wine lees contains both free ceramide and glucosylceramide.

(Example 2: Examination of cleaning process 1)
In this example, the process of washing wine lees, which is a raw material, when obtaining an extract from wine lees was investigated. FIG. 3 shows a method for producing the wine lees extract (“W ceramide extract” in the figure) investigated in this example. On the left side of FIG. 3, W ceramide extract was extracted from wine starch using 95% ethanol (60° C., 6 hours), and then W ceramide extract was further extracted from the residue using 60% ethanol (60° C., 6 hours). 3 hours). In the right part of FIG. 3, wine lees were washed with 50% ethanol (40° C., 1 hour), and then W ceramide extract was extracted from the residue after washing with 95% ethanol (60° C., 6 hours). ). The results are shown in FIGS. 4 and 5. FIG. FIG. 4 shows the content (mg) of each component, and FIG. 5 shows the ratio of each component.

As you can see from this figure, when 95% ethanol extraction is followed by 60% ethanol extraction, many contaminants remain. On the other hand, contaminants could be reduced when the wine lees were first washed with 50% ethanol followed by 95% ethanol extraction.

(Example 3: Examination of cleaning process 2)
In Example 2, it was found that impurities can be removed more effectively by washing the wine lees than by first extracting it with ethanol. Therefore, washing solutions were investigated. In this example, wine lees were washed with water, 10% ethanol, 30% ethanol, or 50% ethanol (both at 40 ° C. for 3 hours), and then 95% ethanol was removed from the residue after washing. was used to extract W ceramide extract (40° C., 6 hours) (FIG. 6). The results are shown in FIG.

As you can see from this figure, when washing with 50% ethanol, we were able to reduce a lot of contaminants, but the amount of ceramide and polyphenol also decreased. Further, when washed with 50% ethanol, the obtained W ceramide extract lost its wine color (not shown). On the other hand, when washed with water, although the amount of contaminants is greater than when washed with 50% ethanol, a large amount of ceramide and polyphenol remain, and the amount of contaminants is also 10% or 30% ethanol. was able to be reduced compared to the case of washing with From this, it was found that water is the most suitable cleaning solution.

(Example 4: Examination of washing temperature)
In Example 3, it was found that washing with water was optimal, so the washing temperature was subsequently investigated. In this example, wine lees were washed with water at 40° C. or 60° C. (both 500 ml, 3 hours), and then W ceramide extract was extracted from the residue after washing with 95% ethanol. (40° C., 6 hours) (FIG. 8). The results are shown in FIG.
As can be seen from this figure, contaminants (indicated by "others" in the figure) were significantly reduced when washed at 60°C compared to when washed at 40°C.

(Example 5: Examination of washing time)
In Example 4, it was found that washing the wine lees with water at 60° C. was optimal, and therefore the washing time was investigated. In this example, wine lees were washed for 30 minutes, 60 minutes, and 180 minutes (500 ml, 60° C. in each case), and then W ceramide extract was extracted from the residue after washing using 95% ethanol ( 40° C., 6 hours) (FIG. 10). The results are shown in FIG.

As you can see from this figure, contaminants were removed most when washed with water for 30 minutes. On the other hand, when washed with water for 60 minutes, the contents of glucosylceramide and free ceramide became the highest while impurities were removed. From this, it was found that the optimal washing time is 60 minutes.

(Example 6: Examination of amount of water added)
In Examples 4 and 5, it was found that washing the wine lees with water at 60° C. for 60 minutes was optimal, so the amount of water added during washing was investigated. In this example, 50 g, 60 g, and 75 g of wine lees were washed with 500 ml of water (10 times water, 8.3 times water, and 6.7 times water, each at 60° C. for 1 hour), Thereafter, W ceramide extract was extracted from the residue after washing using 95% ethanol (40° C., 6 hours) (FIG. 12). The results are shown in FIG.

As can be seen from this figure, when washing with 10 times water (50 g of wine lees, 500 ml of water), 8.3 times water (60 g of wine lees, 500 ml of water), 6.7 times water (75 g of wine lees, water 500 ml), contaminants (indicated by "others" in the figure) could be greatly reduced.

(Example 7: Examination of pH adjustment)
In Examples 2 to 6, when obtaining a ceramide extract from wine lees, it was found that it was optimal to wash the wine lees with 10 times the amount of water at 60 ° C. for 60 minutes. The pH of the W ceramide extract obtained by extraction was examined. In this example, tartaric acid, citric acid, or sodium carbonate was added to the W ceramide extract obtained by ethanol extraction, and then an excipient (cyclodextrin) was added, followed by freeze-drying into powder. The powder color in the case was confirmed (Fig. 14). The results are shown in FIG.

As can be seen from Fig. 15, it was found that it was possible to adjust the color of the powder to a desired color by adjusting the pH of the extracted extract. Especially when the pH was adjusted to 4.0 or 3.5 with citric acid or tartaric acid, a wine-colored powder could be obtained.

(Example 8: Cell proliferation test)
In order to examine the cell proliferation effect of the ceramide-containing composition of the present invention, a cell proliferation test was conducted using human epidermal cells (HaCaT cells). Human epidermal cells (HaCaT cells) were seeded on a 96-well plate, and 24 hours after seeding, they were cultured in a medium containing various concentrations of ceramide extract obtained from wine lees. Cell proliferation was measured after 3 hours of culture. cell proliferation
It was measured using Counting Kit 8 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.). The results are shown in FIG.

As a result, it was found that the ceramide extract obtained from wine lees promotes cell proliferation in keratinocyte cells (HaCaT cells) (see 0.01-1 μg/mL addition group in FIG. 16).

(Example 9: resveratrol amount measurement)
In this example, the amount of resveratrol in the ceramide-containing composition of the present invention was measured. Resveratrol is one of polyphenols with antioxidant action and is known as a component contained in red wine. By ingesting resveratrol, effects such as preventing cell oxidation and improving skin elasticity are expected. In this example, the amount of resveratrol contained in red wine, wine lees (wine crystals), and ceramide-containing composition (wine ceramide) was measured using HPLC. The results are shown in FIG.

As is clear from this figure, the amount of resveratrol contained in the ceramide-containing composition (wine ceramide) of the present invention is significantly increased compared to the amount of resveratrol contained in red wine or wine sediment (wine crystals). It can be seen that

(Example 10: Expression enhancement effect of various gene markers by wine ceramide)
A human keratinocyte cell line (HaCaT) was seeded in a 12-well plate at a cell number of 1×10 ⁵ cells/well and cultured in DMEM medium containing 10% FBS (37° C., 5% CO ₂ ). After overnight culture, the cells were cultured for 72 hours in DMEM medium (FBS-free) supplemented with wine ceramide dissolved in DMSO and ceramides derived from other raw materials (Table 1) at predetermined concentrations (the final concentration of DMSO added was 0). .1%). Ceramides derived from other raw materials were purchased from manufacturers listed in Table 1. Wine ceramides were extracted by the optimized procedure shown in Examples 2-6 (water wash with 10 times water at 60°C for 60 minutes and 95% ethanol extraction at 40°C for 6 hours).

A DMEM medium (FBS-free) supplemented with DMSO to a final concentration of 0.1% was used as a control. After culturing for 72 hours, mRNA was collected from the cells and cDNA was prepared. Using this, the mRNA expression of each marker was measured by qPCR. Each marker evaluated is as follows.
・Differentiation markers; Pro-Filaggrin, Loricrin
・Ceramide synthesis pathway marker; Serine-Palmitoyl Transferase

The results are shown in Figures 18-21. As shown in FIG. 18, wine ceramide was effective in normalizing the differentiation marker Pro-filagrin (a protein that forms the stratum corneum of the skin. A raw material of natural moisturizing factor (NMF)) and Loricrin (a protein that forms the stratum corneum of the skin. protein that serves as a scaffold for formation) increased the mRNA expression level. This indicates that wine ceramide promotes keratinocyte differentiation.

In addition, as shown in FIG. 19, wine ceramide increased the mRNA expression level of the ceramide synthesis pathway marker SPT: Serine-Palmitoyl Transferase (an enzyme of the ceramide de novo synthesis pathway) compared to the control. This indicates that wine ceramide promotes the ceramide synthesis pathway.

FIG. 20 also shows the Pro-filagrin mRNA expression level and SPT mRNA expression level when comparing the wine ceramide of the present invention with glucosylceramide and free ceramide. The amounts of glucosylceramide and free ceramide added were calculated based on the equivalent amounts of glucosylceramide and free ceramide contained in wine ceramide. /mL was used. Glucosylceramide and free ceramide used were those isolated from wine ceramide. As can be seen from this figure, wine ceramide is more effective in increasing the respective mRNA expression levels than glucosylceramide alone or free ceramide alone.

FIG. 21 also shows the Pro-Filaggrin mRNA expression level and SPT mRNA expression when comparing the wine ceramide of the present invention with each ceramide obtained from raw materials other than wine (rice ceramide, pine ceramide, and konjac ceramide). indicated the quantity. The composition of each ceramide is as shown in Table 1. As can be seen from this figure, wine ceramide is more effective in increasing the respective mRNA expression levels than rice ceramide or pine ceramide.

(Example 11: Effect of wine ceramide on keratinocytes)
The efficacy of wine ceramide was evaluated in primary cultured keratinocytes cultured in 3D using Transwell. The same wine ceramide as in Example 10 was used. A culture medium containing wine ceramide was added to each of the stratum corneum side (upper well) and the basement membrane side (bottom well) of the transwell for a predetermined period of time and cultured. As a control group, the cells were cultured in a medium containing 0.1% DMSO. After culturing in each medium, frozen sections of 3D-cultured primary cultured keratinocytes were prepared and subjected to hematoxylin-eosin staining and immunostaining using an anti-ceramide antibody.

The hematoxylin/eosin-stained sections were observed under a phase-contrast microscope, and the results of measuring the thickness of the epidermal layer are shown in FIG. As can be seen from the results, the wine ceramide treatment increases the thickness of the skin. As for skin thickness, it is known that skin turnover decreases and becomes thinner with aging. may have also promoted

Sections immunostained with an anti-ceramide antibody were also observed under a fluorescence microscope, and the results of measuring the ceramide content in the epidermal layer are shown in FIG. As can be seen from these results, in the 3D cultured keratinocytes treated with wine ceramide, the immunostained area is enlarged and the amount of ceramide is increased compared to the control group. As shown in Example 10, in the planar culture (2D culture), the mRNA expression level of the ceramide synthesis pathway marker (SPT) was increased. However, it can be seen that the new synthesis of ceramide increased.

(Note)
While the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure is to be construed in scope only by the claims. The patents, patent applications and other publications cited herein are hereby incorporated by reference in their entirety to the same extent as if the content itself were specifically set forth herein. It is understood. This application claims priority to Japanese Patent Application No. 2021-169620 filed on October 15, 2021 and Japanese Patent Application No. 2022-94514 filed on June 10, 2022 with the Japan Patent Office. , the contents of which are hereby incorporated by reference as though they constitute the subject matter of this application in their entirety.

The present invention can provide a ceramide-containing composition derived from wine fermentation lees. Moreover, according to the method of the present invention, it is possible to stably extract a ceramide composition from wine fermentation lees while removing contaminants, and to control the color of the ceramide composition. Furthermore, the ceramide composition of the present invention contains free ceramide and uses highly safe natural materials such as wine fermented lees. It is useful in fields such as cosmetics, pharmaceuticals, and quasi-drugs because it has excellent safety.

Claims

A cosmetic and/or oral composition containing polyphenols and ceramides.
The composition according to claim 1, wherein the ceramide comprises free ceramide and glucosylceramide.
The composition according to claim 2, wherein the composition contains at least about 1% by weight of free ceramide.
The composition according to claim 2 or 3, wherein said composition comprises at least about 1% by weight of glucosylceramide.
The composition according to any one of claims 1 to 4, wherein the composition contains at least about 1% by weight of polyphenols.
of claims 1-5, wherein the weight ratio of free ceramide, glucosylceramide, and polyphenol in the composition is about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. A composition according to any one of the preceding claims.
The composition according to any one of claims 1 to 6, wherein the weight ratio of free ceramide, glucosylceramide and polyphenol in the composition is about 1.0: about 1.0: about 1.0.
The composition according to any one of claims 1 to 7, wherein the composition has a pH of less than about 5.
The composition according to any one of claims 1 to 8, comprising at least about 10 mg resveratrol per about 100 g.
The composition according to any one of claims 1 to 9, which is for food.
The composition according to any one of claims 1 to 9, which is used for cosmetics.
The composition according to any one of claims 1 to 11, wherein the polyphenol and the ceramide are derived from wine fermentation lees.
The composition according to any one of claims 1 to 12, for promoting keratinocyte differentiation.
The composition according to any one of claims 1 to 13, for promoting ceramide synthesis.
A ceramide-containing composition derived from wine fermentation lees.
The composition according to claim 15, wherein the ceramide-containing composition contains at least about 1% by weight of free ceramide.
The composition according to claim 15 or 16, wherein said ceramide-containing composition comprises at least about 1% by weight of glucosylceramide.
The composition according to any one of claims 15 to 17, wherein the ceramide-containing composition contains at least about 1% by weight of polyphenols.
of claims 15-18, wherein the weight ratio of free ceramide, glucosylceramide, and polyphenol in the composition is about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. A composition according to any one of the preceding claims.
20. The composition of any one of claims 15-19, wherein the weight ratio of free ceramide, glucosylceramide, and polyphenol in the ceramide-containing composition is about 1.0: about 1.0: about 1.0. thing.
The composition according to any one of claims 15 to 20, wherein the ceramide-containing composition has a pH of less than about 5.
The composition according to any one of claims 15-21, comprising at least about 10 mg resveratrol per about 100 g.
The composition according to any one of claims 15 to 22, which is for food.
The composition according to any one of claims 13 to 22, which is used for cosmetics.
The composition according to any one of claims 15 to 24, for promoting keratinocyte differentiation.
The composition according to any one of claims 15 to 24, for promoting ceramide synthesis.
A method for producing a ceramide-containing composition, comprising:
A step of washing the wine fermented lees;
and extracting the residue obtained by said washing with a polar solvent.
The method according to claim 27, wherein the polar solvent comprises ethanol.
The method according to claim 28, wherein the ethanol concentration is about 95%.
The method according to any one of claims 27 to 29, wherein the washing step is performed with water.
The method according to any one of claims 27 to 30, further comprising a step of adjusting the pH of the extract obtained by the extracting step.
The method of claim 31, wherein said pH adjustment is performed with tartaric acid or citric acid.