JP4883449B2 - Ceramide-containing composition and method for producing the same - Google Patents

Description

  The present invention relates to a composition containing ceramides extracted from tea leaves and the production thereof, and more particularly to a composition containing ceramides that can be extracted by utilizing tea husk as waste and the production thereof.

  Ceramides are complex lipids known to exist widely in animals and plants, and include ceramides composed of sphingosine and fatty acids, and glycosylceramides in which fatty acids and sugars are added to sphingosine, so-called cerebrosides. As plant-derived ceramides, it is known that glycosylceramide mainly exists.

  Ceramides are constituent lipids of the skin and are used as a cosmetic material for external use. In recent years, it has been confirmed that oral intake has an effect on the moisture retention of skin, such as improvement of dry skin and improvement of fine wrinkles (Non-Patent Document 1), and it is used. For oral intake, materials with higher safety are required, but until now, ceramides have been used from animal tissues, especially from bovine brain. Because of the possibility of infection, highly safe plant-derived ceramide materials have been sought and used.

  Grains and moss are often used for the production of plant-derived ceramide materials, and in particular, rice (Non-patent Document 2, Patent Document 1), wheat (Non-patent Document 3), soybean (Non-patent Document 4, Patent documents 2) and those derived from straw (Patent document 3) have been reported. However, wheat and soy, which are most commonly used as raw materials for ceramides, are considered to be specific raw materials for allergy labeling, and rice is generally known to cause atopic dermatitis. At present, even in the case of plant-derived ceramides, a more safe raw material is required.

The content of ceramides per extraction raw material is very small and is estimated to be about 0.01% to 0.05% (Patent Documents 1, 2, 3 and Non-Patent Documents 2 and 3). Since raw materials are required and the residue after extraction increases, a high content of ceramides and an inexpensive extraction material have been demanded.

On the other hand, tea has no reports of allergies due to eating, and is considered to be a highly safe extraction material. In addition, tea leaves are used in large quantities for drinking, and in recent years, with the increasing demand for tea cans in commercial cans and PET bottles, a large amount of tea leaves are discharged from the factory and are produced annually for the production of tea beverages. About 18,000 tons of crude tea are used (see Non-Patent Document 5), and it is estimated that about 75,000 tons of tea leaves are discharged. Most of the discharged tea husk is processed as fertilizer or waste (see Non-Patent Document 6), and in the future, a means of using the tea husk with higher added value is desired.

The tea husk is a food waste and has no value as a food. Compared with the ceramide extract materials derived from rice, wheat, soybeans, and rice bran currently used, tea husk is easy to obtain in large quantities from tea beverage manufacturing plants, and industrial ceramides It is considered to be a suitable material for the manufacture of
Although there is a description of extraction of ceramides using oolong tea as teas (Patent Document 4), here, a solvent for direct extraction is added without pretreatment of tea leaves, and ceramides in tea leaves are added. Has not been extracted efficiently and sufficiently. So far, there has been no example of using the tea husk after extracting the beverage as an extraction material for ceramides.

JP-A-11-279586 Japanese Patent No. 2119895 Japanese Patent No. 3650587 Japanese Patent Laid-Open No. 2003-221592 Fragrance Journal, 23 (1), 81-85 (1995) Agric.Biol.Chem., 49,2753-2762 (1985) Agric. Biol. Chem., 49, 3609-3611 (1985) Chem. Pharm. Bull., 38 (11), 2933-2938 (1990) “Demand for cereals and tea: trends and prospects”, Ministry of Agriculture, Forestry and Fisheries General Food Bureau (2003) "Itoen Environmental Report 2003"

The present invention aims to develop a highly safe new ceramide material that does not use a specific raw material for allergy labeling, focusing on tea husks that have not been noticed so far, and using it as an extraction raw material. .

As a result of intensive studies, the present inventors have found that tea leaves contain an amount of ceramide that is equal to or greater than rice, wheat, and rice that have been used as ceramide materials so far. It was clarified and found that it can be easily extracted with an organic solvent, particularly an alcohol that can be used as a food. Moreover, when using as a foodstuff, the ceramide containing composition with higher safety | security was able to be obtained among plant-derived ceramide raw materials, without using the specific raw material of an allergy display by using a tea leaf as an extraction raw material.

Furthermore, focusing on the fact that ceramides are sparingly soluble in water, it has been found that many ceramides are left in the tea husk, which is a waste product after extraction of tea leaves with water or the like. By extracting ceramides using this tea husk, it was found that ceramides can be obtained more efficiently than directly extracting from tea leaves not extracted with water, and the present invention as described below was completed. .
(1) A composition containing ceramides obtained by water-extracting a raw leaf of a plant belonging to chanoki (scientific name: Camellia sinensis (L.) O. Kuntze) or a processed tea leaf and extracting the residue with an organic solvent. Its manufacturing method.
(2) The ceramide-containing composition according to (1) and a method for producing the same, wherein the residue obtained by water extraction of the processed tea leaves is a tea shell of tea for beverages.

The ceramide-containing composition of the present invention consists of an extract obtained by extracting a tea leaf with an organic solvent from an extraction residue obtained by extracting water with hot water or hot water. Also, ceramide-containing compositions extracted from tea leaves are less likely to develop allergies, unlike ceramide-containing compositions derived from rice, wheat, and soybean, which are commonly used as raw materials for extraction. It can be said that this is a high ceramide material.
Further, the present invention is a ceramide-containing composition and a method for producing the same, which are excellent in that tea leaves, which are tea leaf residues used for dim sum, can be used as a suitable raw material and waste can be utilized.

The plant belonging to the tea tree (scientific name: Camellia sinensis (L.) O. Kuntze) in the present invention is a Chinese species (scientific name: C. sinensis var. Sinensis) that is a raw material for green tea and oolong tea, and a raw material for black tea. Assam species (scientific name: C. sinensis var. Assamica), and other varieties. The tea leaves preferably used as the raw material of the present invention may be any raw leaves of plants belonging to the tea tree, which are usually used as various dim sum tea raw materials, or teas processed from raw tea. Teas obtained by processing fresh tea refer to non-fermented tea, semi-fermented tea and fermented tea produced by a known method. Examples include Japanese tea (sencha, bancha, deep-mushroom sencha, stem tea, gyokuro, brown rice tea, matcha tea, guricha, hojicha, etc.), Chinese green tea, blue tea (oolong tea), black tea, black tea, etc. Non-fermented tea, semi-fermented tea, fermented tea may be used singly or as a mixture, and blended tea mixed with tea derived from other plant tissues is also included. Particularly preferably, Japanese tea can be used (hereinafter, the above-mentioned fresh leaves and teas are collectively referred to as tea leaves).
The tea husk refers to a residue obtained by extracting the above teas with hot water or water. Of course, the tea husk may be a residue using tea as dim sum.

  In the present invention, when water is extracted from tea leaves, water at 10 ° C. or higher, preferably 60 ° C. or higher, more preferably 80 ° C. or higher, is used for 3 seconds to 2 days, preferably 5 seconds to 120 minutes, Preferably, it is a residue obtained by extraction treatment for 10 seconds to 30 minutes. It should be noted that the water to be extracted does not have to be completely pure water, and even if it contains salt like seawater, or alcohol such as sugar or alcohol, or acetic acid is added, it is considered to be a substantially aqueous solution. Anything can be used. The amount of the solvent used for extraction is 1 to 10,000 parts by weight, preferably 1 to 1,000 parts by weight, and more preferably 5 to 50 parts by weight per 1 part by weight of the dried tea leaf raw material. The residue after the water extraction treatment can be recovered from the extract by a known method. For example, it may be filtered using cloth, paper, wire mesh or the like, decanted, centrifuged, or the like. The residue generated after water extraction of tea leaves may be a residue obtained by extracting processed tea leaves for drinking tea with hot water or hot water in a factory or home, so-called tea husk. In particular, tea husks produced industrially in the production process of tea beverages are suitable in that a large amount of tea husks having a uniform quality can be obtained.

  The ceramide-containing composition in the present invention is obtained by extracting the residue after the above-described tea leaves are extracted with water (hereinafter referred to as “tea husk”) using an organic solvent. The solvent that can be used for extraction is not particularly limited as long as it can efficiently extract the components in the tea husk. For example, lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, etc., or propylene glycol , Polyhydric alcohols such as 1,3-butylene glycol, acetone, methyl ethyl ketone, butyl methyl ketone, acetic acid, ethyl acetate, acetonitrile, diethyl ether, dioxane, dichloromethane, trichloroethylene, carbon tetrachloride, chloroform, propane, butane, pentane, hexane And lower hydrocarbons such as benzene, toluene, xylene, aniline, pyridine, pyrrolidone and the like. These solvents may be used alone or in combination of two or more, or may be an organic solvent containing water. When the above lower alcohols and polyhydric alcohols are used as hydrous alcohols, the water content is 30% by mass or less, preferably 20% by mass or less, more preferably 10% by mass or less. As the extraction solvent, alcohols and mixed solvents thereof are preferable, and ethanol and hydrous ethanol having a water content of 10% by mass or less are particularly preferable.

  The extraction conditions may be appropriately selected depending on the solvent species. When the extraction solvent is ethanol or hydrous ethanol, it is 10 ° C. or higher, preferably 20 ° C. or higher, 1 minute to 100 hours, preferably 3 minutes to 50 hours, more preferably 5 It is desirable to extract from minutes to 20 hours. The amount of the organic solvent used for extraction is 1 to 10,000 parts by weight, preferably 1 to 1,000 parts by weight, more preferably 5 to 50 parts by weight per 1 part by weight of the dried tea leaf raw material. In addition, although it is not necessary to dry raw material tea shells in particular, it is necessary to be careful because the moisture concentration in the extraction solvent increases if it contains too much moisture. The residue after the extraction treatment can be separated from the extract by a known method. For example, it may be filtered using a cloth, paper, wire mesh or the like, decanted or centrifuged. The extract thus obtained can be obtained by removing the solvent by a conventional method. This extract is the ceramide-containing composition of the present invention. Solvent removal can be shortened by heating, but it is desirable that the heating temperature does not exceed 120 ° C. in order to prevent partial decomposition and transpiration of ceramides. Furthermore, concentration drying under reduced pressure is preferable because it not only prevents the alteration of the extract but also can be processed in a short time. The solvent used in the extraction can be separated from the extract and then used again in the extraction. For example, ethanol and other materials often contain moisture in the raw tea shells. Use it.

The obtained ceramides-containing composition can be used after purification of ceramides by a conventional method. The method for purifying ceramides is not particularly limited, and extraction and washing operations using various solvents, liquid-liquid distribution methods, chromatography methods using various carriers such as silica gel and resins, and crystal methods can be used.

The quantification of ceramides in the ceramide-containing composition can be analyzed by a known method. For example, a thin layer chromatography method, a high performance liquid chromatography method, a gas chromatography method and the like can be mentioned. Particularly conveniently, ceramides can be quantified by thin layer chromatography. Using a standard product, convert the spot obtained by silica gel thin-layer chromatography using commercially available ceramides or high-purity purified products as standard, using image processing software or a densitometer, etc. By using the calibration curve, quantitative analysis can be performed.

The ceramide-containing composition obtained as described above can be used after being processed into a tablet, capsule, emulsion, gel, powder using a carrier, or the like by a conventional method. Moreover, you may add and use for various foodstuffs, feed, a pharmaceutical, cosmetics, etc. In taking the above-mentioned form, a fragrance, a pigment, oils and fats, alcohols, a moisturizer, an ultraviolet absorber, an antioxidant, an antiseptic, a surfactant, and the like can be blended as appropriate according to the purpose.

EXAMPLES Next, although an Example demonstrates this invention in more detail, this invention is not restrict | limited to these Examples at all.
Example 1
Using 20 g of commercially available bancha as a raw material, 300 ml of demineralized water was added, heated to 80 ° C., stirred and extracted for 30 minutes, and then the supernatant and residue were separated by filtration. The collected supernatant was concentrated under reduced pressure to obtain 6.3 g of a hot water extract (referred to as Extract A). This process is called a water extraction process.
Next, as an organic solvent extraction step, the residue after the hot water extraction (tea husk) was dried on a filter paper at room temperature for 1 hour, and then 99.5% ethanol 300 ml was added, followed by stirring and extraction at room temperature for 16 hours. . The extract and residue were separated by filtration. The extract was concentrated under reduced pressure at room temperature to obtain 2.16 g of an ethanol extract (referred to as Extract B).

(Example 2)
Using 10 g of commercially available black tea as a raw material, 0.56 g of an ethanol extract (referred to as Extract C) was obtained through a water extraction step and an organic solvent extraction step.

(Example 3 (Comparative Example 1))
Using 20 g of commercially available bancha as a raw material, 300 ml of 99.5% ethanol was added, followed by stirring and extraction at room temperature for 16 hours. The extract was concentrated under reduced pressure to obtain 1.31 g of an ethanol extract (referred to as Extract D).

Example 4
Next, ceramides in the obtained extract were purified.
First, extract B is prepared in a 1% (W / V) ethanol solution, 0.4% (W / V) of activated carbon is added thereto, and the absorbance (OD664nm) is 0.1 or less at room temperature. The mixture was stirred to remove lipophilic colors as impurities. From the supernatant obtained by removing the activated carbon by filtration, the solvent was removed under reduced pressure to recover 1.74 g of a solid content. Thereto, hexane was added and dissolved so that the solid content concentration became 1.8% (W / V), and then the same amount of 60% (V / V) ethanol aqueous solution was added and stirred vigorously, and then static The hexane layer containing the complex lipid was recovered by separating into two layers. The solvent of the collected hexane layer solution was removed under reduced pressure to obtain 0.754 g of a solid content from which hydrophilic impurities were removed. 0.5N methanolic potassium hydroxide was added so that the obtained solid content would be 10% (W / V), and the mixture was shaken at 37 ° C. for 1 hour with 140 rotations to obtain complex lipids other than ceramides. Decomposition was performed. After decomposing, neutralize with methanolic 3N hydrochloric acid, add 1/10 volume of 20% aqueous sodium sulfate solution and 2 volumes of water, and then extract three times with the same volume of chloroform. . The obtained chloroform extract was dehydrated with anhydrous sodium sulfate, and then the solvent was removed under reduced pressure to recover the solid content. The collected solid content was washed with a small amount of acetone, dried under reduced pressure, and chromatographed and purified using a silica gel column. A sample for separation is dissolved in a small amount of chloroform, introduced into a column packed with silica gel (inner diameter 10 mm, length 200 mm), and eluted sequentially with a chloroform-methanol mixed solution while changing the mixing ratio from 40: 1 to 1: 1. To obtain 9.1 mg of a fraction containing the desired ceramides (referred to as purified product E). Purified product E was detected as a single spot by thin layer chromatography on silica gel. The analysis conditions for thin layer chromatography were as follows: a sample was spotted on a silica gel plate, and then a chloroform / methanol / water mixture of 65 to 16 to 2 (volume ratio) was developed. Subsequently, spots were detected by spraying the plate with 50% (V / V) aqueous methanol solution containing 10% (W / V) α-naphthol, followed by heating with 50% sulfuric acid (TLC analysis). It is called condition 1.)

Next, the purified product E was tableted with potassium bromide (KBr) crystals, and the result of infrared absorption spectrum analysis is shown in FIG.
Absorption of 1539 and 1638 derived from acylamide bonds of ceramides and absorption of 3337 and 1080 derived from sugar were confirmed, and it was found that ceramides derived from tea husks were glycosylceramides.

(Example 5)
The molecular structure of the purified product E by strong acid decomposition was confirmed.
6.0 mg of the purified product E was suspended in 1.5 ml of methanolic 2N hydrochloric acid and subjected to decomposition treatment at 70 ° C. for 5 hours. After the decomposition treatment, the mixture was neutralized with methanolic 0.5N potassium hydroxide, and the total volume was made 9 ml with methanol (referred to as decomposition solution F). Using the obtained decomposition solution F, saccharides, sphingosine and fatty acids contained therein were quantified.
For analysis of saccharides in the decomposition solution F, first remove the solvent of the decomposition solution F under reduced pressure, then suspend in the same amount of water, filter through a membrane filter, and then perform qualitative analysis by thin layer chromatography and high performance liquid. Chromatographic quantitative analysis was performed. The analysis conditions of thin-layer chromatography of saccharides were as follows: after spotting a sample on a silica gel plate, 1-butanol, pyridine, and water were developed in a mixed solution of 60:25:15, and after spraying a naphthoresorcin sulfate solution, Upon heating, sugar spots were detected. The conditions for quantitative analysis of saccharides by high performance liquid chromatography were as follows: GELPACK GL-C610, 10.7 mm i. d. x 300 mm (manufactured by Hitachi), the column temperature is 60 ° C., the mobile phase is distilled water, and the flow rate is 1.0 ml / min. The detection was performed by differential refraction.
The sphingosine in the decomposition solution F was quantified by colorimetrically determining the sphingosine produced by the decomposition as a methyl orange complex (Biochemical Experimental Method 9 Introduction to Lipid Analysis, Yasuhiko Fujino, Society Publishing Center (1978), p. 143). . The solvent was removed under reduced pressure using 0.1 ml of decomposition solution F, 1 ml of methanolic 2N hydrochloric acid and 0.5 ml of 7N sodium hydroxide were added, and the total amount was adjusted to 2 ml with water. To this solution, 5 ml of ethyl acetate was added, followed by extraction with stirring. After removing the aqueous layer formed by standing, washing with 2 ml of water was performed twice. Thereafter, 2 ml of 0.01 M acetate buffer solution and 0.1 ml of methyl orange solution were added, stirred vigorously and allowed to stand, and the absorbance (415 nm) of the resulting ethyl acetate layer was measured with an absorptiometer. As a standard, the same operation was performed with a commercially available sphingosine, a calibration curve was prepared, and the amount of sphingosine in the degradation product was quantified.
Analysis of the fatty acid in the decomposition solution F was performed by quantifying the total amount of fatty acid by measuring the carboxylic acid of the fatty acid by titration (Biochemical Experimental Method 9 Introduction to Lipid Analysis, Yasuhiko Fujino, Society Publishing Center (1978), p. 140- ). Decomposition solution F 8 ml of solvent was removed under reduced pressure, then dissolved in 6 ml of a propylene glycol solution containing 6.5% (W / V) sodium hydroxide, and treated at 70 ° C. for 2 hours for decomposition. . Thereafter, 2 ml of water and 2 ml of diethyl ether were added and stirred, and allowed to stand to collect a two-layered aqueous layer. The recovered aqueous layer was adjusted to pH 1-2 with 5N sulfuric acid aqueous solution, extracted with 2 ml of diethyl ether three times, and the recovered diethyl ether layer was washed twice with a small amount of water and then anhydrous sodium sulfate. And then titrated with methanolic 0.02N sodium hydroxide.

  From the results of the above analysis, it was confirmed that the decomposition solution F of the purified product E with strong acid contained glucose, sphingosine and fatty acid in a molar ratio of 1: 1.0 to 0.9. From this, it was confirmed that the purified product E was monoglucosylceramide.

(Example 6)
The amount of glucosylceramide contained in the extracts A, B, C and D was quantified by silica gel thin layer chromatography using the purified product E as a standard. After spotting a sample on a silica gel alumina plate, the spot was detected under TLC analysis condition 1 of Example 4. The spot-detected plate is converted to a digital image by a scanner, and the glucosylceramide spots are digitized by image processing software (Scion Image). The glucosyl contained in the extract is based on a calibration curve created using a standard. Ceramide was quantified.

  From Table 1, it was confirmed that the extract A, which is a tea beverage, did not contain glucosylceramide at all, and the extract B, which is an extract of tea shells, contained glucosylceramide. In addition, it was confirmed that glucosylceramide of the same degree as bancha can be extracted even using black tea leaves that are fermented tea leaves. On the other hand, it was found that glucosylceramide in tea leaves could not be efficiently extracted with Extract D, which was directly extracted with ethanol from tea leaves that were not extracted with water. From the above results, it was proved that extraction of ceramides using tea leaves after water extraction treatment of tea leaves was efficient.

Since the ceramide-containing composition of the present invention is produced using a material produced in the food production process, it is highly safe and can be suitably used as a food, particularly a functional food, a cosmetic, and a pharmaceutical. .

FIG. 1 shows the result of measuring the infrared absorption spectrum of purified product E tableted with potassium bromide (KBr). (Example 5)

Claims (2)

It is obtained by water extraction of fresh leaves or processed tea leaves belonging to chanoki (scientific name: Camellia sinensis (L.) O. Kuntze) at 10 ° C. or more for 3 seconds to 2 days, and extracted from the residue with an organic solvent. A method for producing a ceramide-containing composition. The method for producing a ceramide-containing composition according to claim 1, wherein the residue obtained by water-extracting the processed tea leaves at 10 ° C or more for 3 seconds to 2 days is a tea shell of tea for beverages.

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