JP7245966B2 - Sphingosine Derivatives Adsorbing AGEs - Google Patents

Description

The present invention relates to a sphingosine derivative exhibiting an AGEs-adsorbing action.

AGEs is an abbreviation for Advances Glycation End Products, and is called Advanced Glycation End Products or Advanced Glycation End Products. It is a complex produced by the in vivo reaction of proteins and carbohydrates, and is a substance that is expressed in various forms of aging in the body.

AGEs are indicators of aging and physiological reactants that induce aging. Reducing these AGEs is a significant strategy for preventing aging. Therefore, various studies are being conducted to reduce AGEs.

As an invention related to AGEs, for example, there is an invention of a protein saccharification reaction inhibitor containing a black galingal extract as an active ingredient. Reference 1).

In addition, there is an invention of an anti-glycation composition characterized by containing either one of sugarcane stem leaf powder or sugarcane stem leaf extract and either one of barley leaf powder or barley leaf extract. However, the structure has not been specified, and its industrial application is limited (see, for example, Patent Document 2).

JP 2017-210452 Patent No. 6260804

The AGEs adsorption action by existing substances is mild, and there is a problem that their industrial use is limited, and chemically synthesized substances have safety problems and their use is limited.

Therefore, there is a demand for a natural product that is highly safe and exhibits excellent AGEs adsorption action.

In order to achieve the above object, the invention described in claim 1 relates to a sphingosine derivative having AGEs-adsorbing action represented by the following formula (1).

Since the present invention is constructed as described above, it has the following effects.

The sphingosine derivative according to claim 1 is excellent in AGEs adsorption action.

Embodiments embodying the present invention will be described in detail below.

A sphingosine derivative exhibiting an AGEs-adsorbing action has a structure represented by the following formula (1).

A sphingosine derivative exhibiting an AGEs-adsorbing action as shown in the above formula (1) is composed of one sphingosine molecule, one pyridine molecule having two carboxylic acid molecules, and three guluronic acid molecules. All of these molecules and their bonds are natural types existing in nature, and the bonds between each molecule are linked by ester bonds, peptide bonds or ether bonds found in nature.

This sphingosine derivative can be obtained by chemical synthesis using sphingosine, carboxylic acid, pyridine and guluronic acid as raw materials. However, in this case, chemical synthesis involves a large loss of raw materials and increases production costs, which limits industrial use.

On the other hand, chemically synthesized high-purity sphingosine derivatives are used to obtain analytical standards and trace samples.

It is preferable to analyze the structure of this sphingosine derivative because the active ingredient can be identified. It is also preferable because it can be used as an indicator of the content of active ingredients when used in products and formulations for sale.

When analyzed by H-NMR at 500 MHz in deuterated chloroform using a chemically synthesized high-purity (purity 98.2%) standard for structural analysis of this sphingosine derivative, the peak position was 0.95, 1.00, 1.65, 1.66, 1.75, 1.77, 1.79, 1.84, 1.96, 2.08, 2.52, 2.56, 2.75, 2. 77, 3.30, 3.31, 3.32, 3.42, 3.61, 3.67, 3.69, 3.74, 3.91, 3.93, 3.97, 4.02, Found at 4.06, 4.23, 4.84, 4.86, 5.05, 5.99, 6.02, 6.14, 6.17, 7.55 and 7.58 ppm.

In addition, when analyzed by C-NMR at 500 MHz in deuterated chloroform as structural analysis of this sphingosine derivative, the peak positions are 13.2, 13.5, 14.3, 16.4, 32.4, and 32. .8, 35.7, 41.4, 41.5, 42.0, 42.9, 44.5, 48.6, 52.4, 63.3, 63.5, 67.0, 67.7 , 72.2, 72.3, 75.4, 75.5, 78.6, 78.7, 79.1, 79.3, 80.6, 95.5, 95.8, 101.3, 101 .6, 110.3, 111.5, 124.6, 125.4, 131.7, 133.1, 154.7, 155.9, 169.9, 170.0, 174.5 and 175.3 ppm recognized by

Further, it is preferable that this sphingosine derivative is also analyzed by high-performance liquid chromatography or the like, and the respective analytical data are aggregated to identify its structure. Its chemical formula is C43H62N2O22.

That is, it is composed of 43 carbon atoms, 62 hydrogen atoms, 2 nitrogen atoms and 22 oxygen atoms. In addition, the sphingosine derivative, which is the constituent component, is a naturally occurring compound.

Originally, this sphingosine is a major component of highly fat-soluble sphingolipids or sphingolipids widely distributed in animals, plants and microorganisms, and has a fatty side chain, two hydroxyl groups and an ammonium group. It is also widely present as a component of cell membranes, nerve myelin sheaths, and ceramides. The state in which sphingosine is activated is related to the state in which the cell membrane is stably maintained.

In this sphingosine derivative, two hydroxyl groups of one molecule of sphingosine form an ester bond with two carboxylic acids bonded to one molecule of pyridine ring. Since the pyridine ring exhibits weak basicity, it is electrically positive, that is, in a positive state. On the other hand, the ammonium group of sphingosine forms a peptide bond or an ester bond with one molecule of guluronic acid. In addition, three molecules of guluronic acid are linked by ether bonds as carbohydrates. Both of the carboxylic acids of the two molecules of guluronic acid are free forms and exhibit weak acidity.

It is electrically negative, that is, in a negative state. The fact that the pyridine ring is electrically positive and the guluronic acid is electrically negative has the effect of adjusting the potential of the cell membrane between positive and negative when this derivative is incorporated into the cell membrane. It contributes to the stabilization and activation of cell membranes in cardiomyocytes, muscle cells and skin cells. Furthermore, in terms of affinity, sphingosine exhibits lipophilicity, and pyridine ring and guluronic acid are amphipathic, exhibiting hydrophilicity, and work to maintain the structure of cell membranes.

This sphingosine derivative is highly absorbed through the skin and intestinal tract as a result of its electrical activity and amphipathic nature. In particular, percutaneous absorption increases about four times as much as sphingosine alone.

Furthermore, this sphingosine derivative has a function of adsorbing AGEs. This is because the space between gluconic acid and sphingosine in this sphingosine derivative has the function of adsorbing AGEs. That is, it can bind to the oxidized carbohydrate-protein binding site.

AGEs bound to this sphingosine derivative are phagocytosed and degraded by phagocytic cells of the macrophage system. AGEs disappear by being decomposed. AGEs bound to this sphingosine derivative are also decomposed by proteases in the body. For example, it is degraded by trypsin in blood. Trypsin forms a conjugate with antitrypsin in blood, and antitrypsin is released by contact with AGEs, and AGEs are decomposed by activation of trypsin.

This sphingosine derivative also exhibits adsorption to AGEs receptors on the surface of cell membranes. Since the cell membrane receptor is a protein, it loses its function as a receptor when converted to AGEs. In addition, this sphingosine derivative has the function of being incorporated into cell membranes and stabilizing the cell membranes. It also activates receptors present on the cell membrane. Furthermore, it enhances the fluidity of the cell membrane and enhances the function of the cell membrane. This sphingosine derivative moves into cells, adsorbs AGEs, and excretes them outside the cells.

Furthermore, since the carboxylic acid portion of sphingosine is charged to be weakly acidic, it is preferable that sphingosine has strong acid resistance, exhibits resistance to gastric acid when orally ingested, and increases absorption rate. By stabilizing this sphingosine derivative in the stomach, AGEs contained in meals are adsorbed and excreted.

Moreover, since it is weakly acidic, it is preferable that it does not irritate the skin when applied to the skin. This sphingosine derivative becomes stable even on the surface of the skin, and adsorbs and excretes AGEs adhering to the skin. In subcutaneous tissue, this sphingosine derivative binds to AGEs and causes phagocytic cells such as Langerhans cells to decompose AGEs.

Furthermore, this sphingosine derivative inhibits the formation of AGEs by all substances such as active oxygen, free radicals, ultraviolet rays, chemical substances, side effects of pharmaceuticals, metals and aging, and also adsorbs AGEs.

In addition, since this sphingosine derivative exhibits both fat-soluble and water-soluble properties, it easily passes through animal cell membranes and plant and yeast cell walls and is absorbed into cells. Moreover, it is preferable that a wide range of solvents can be used because it dissolves in both water-soluble solvents and oil-soluble solvents.

Furthermore, it easily passes through the corneocyte membrane of the skin, and maintaining the barrier function of the stratum corneum is preferable from the viewpoint of skin health and beauty. Moreover, this sphingosine derivative is preferable because it passes through cell membranes and adsorbs AGEs in skin cells to promote regeneration and function of cells.

For plants, this sphingosine derivative passes through the cell wall and cell membrane of the plant and enters the plant cell, promoting the adsorption of AGEs, flowering, fruiting, and leaf growth, thereby extending the life of the plant. is preferred. That is, it works as a plant activator.

Moreover, when this sphingosine derivative is powdered, it reacts with a water-soluble solvent to generate hydrogen gas and eliminate active oxygen. The amount of hydrogen gas generated is a saturated concentration of 1.6 ppm, and is generated for about 1 minute to 2 hours after dissolution. Hydrogen gas has a function of scavenging hydroxyl radicals, and has been confirmed to have an excellent active oxygen scavenging action.

In addition to adsorbing AGEs, this sphingosine derivative preferably promotes skin cell functions by promoting cell growth and ceramide, collagen and elastin production.

Nerve cells also adsorb and excrete intracellular AGEs. Nerve cells are susceptible to genetic damage due to reactive oxygen species and amyloid β protein in dementia, Alzheimer's disease, etc., and have the weakness that genes are difficult to repair. Therefore, adsorption of AGEs by this sphingosine derivative is preferable for the purpose of restoring nerve functions and preventing and recovering from neurological diseases.

It is also desirable to protect the myelin sheath and promote the release of neurotransmitters from nerve endings to enhance neurotransmission. Furthermore, the generated hydrogen gas is a low-molecular substance that can pass through the blood-brain barrier and repair damaged brain cells.

It is desirable to increase muscle contraction and increase nerve and muscle activity by increasing the release of acetylcholine from the nerve endings of motor neurons.

In addition, it is preferable that this sphingosine derivative exhibits AGEs-adsorptive action on skin cells and protects the genes of collagen and elastin to increase their production. It is preferable because it increases the use as a cosmetic.

In myocardial infarction, this sphingosine derivative preferably exerts a cardiotonic action by activating cardiac activity through AGEs adsorption to myocardial cells even in coronary artery infarction or ischemia. In addition, hydrogen gas generated at the same time improves damage of active oxygen caused by ischemia-reperfusion in the myocardium.

In particular, this sphingosine derivative promotes angiogenesis, improves blood flow, and lowers blood pressure in blood vessels at the site of infarction.

In addition, this sphingosine derivative promotes the transport of fat to muscle cells during exercise by athletes and the general public, and when people want to build muscle. This is preferred because it is through pathways that activate energy production at the genetic level. In addition, hydrogen gas generated from this derivative during muscle activity eliminates active oxygen during exercise, which is preferable because it reduces genetic damage to muscle cells caused by active oxygen.

This sphingosine derivative is decomposed in vivo by esterase and oxidase in kidney and liver and excreted in urine. It is decomposed into its highly safe constituents sphingosine, carboxylic acid, pyridine and guluronic acid. Therefore, this sphingosine derivative does not accumulate in the body, is decomposed by in vivo enzymes, and is highly safe because the decomposed product is a natural product.

Furthermore, since the sphingosine moiety has a plant-activating action that promotes the growth of plants, the fact that this sphingosine derivative can also promote the growth of plants is preferable from the viewpoint of industrial application.

In addition, when plants are infected with bacteria or viruses, their genes may be damaged. It is preferable to activate AGEs adsorption against such genetic disorders.

This sphingosine derivative also exists in nature, and is found in very small amounts in marine microorganisms such as Sphingomonas and seaweeds such as kelp.

It is possible to extract this sphingosine derivative from the above plants by purification. However, the purification requires a large amount of raw materials and uses organic solvents, etc., which limits industrial use.

This sphingosine derivative is preferably produced by fermenting Sphingomonas and kelp. As a fermentation method, it is obtained by fermenting with Sphingomonas. Since the bacteria used are edible, they are highly safe.

The kelp used as the raw material for this sphingosine derivative is preferable because it has been eaten, is safe, and produces a large amount of the sphingosine derivative. In particular, a sphingosine derivative obtained by purifying a fermentation broth obtained by fermenting Laminaria japonica with Sphingomonas is more preferable because it is produced by a fermentation process using only natural products as raw materials, so that a highly safe sphingosine derivative can be obtained. In addition, by using a sphingosine derivative produced by a production process of purifying a fermentation liquid obtained by fermenting Laminaria japonica with Sphingomonas, it is possible to obtain a sphingosine derivative by a highly safe production method.

When the obtained sphingosine derivative is used as a pharmaceutical material, it is preferable to purify the target sphingosine derivative from the viewpoint of increasing the purity of the target sphingosine derivative and removing impurities.

As pharmaceuticals, it is used as injections, oral agents, or parenteral agents such as ointments, and as quasi-drugs, it is used in tablets, capsules, drinks, soaps, ointments, gels, toothpastes, etc. be.

Oral preparations include tablets, capsules, powders, syrups, drinks and the like. When mixed with the above tablets and capsules, it can be used together with binders, excipients, swelling agents, lubricants, sweeteners, flavoring agents and the like. Such tablets may also be coated with shellac, sugar or the like.

In addition, in the case of the above-mentioned capsules, the above-mentioned materials may further contain a liquid carrier such as oils and fats. Sweeteners, preservatives, dyes, flavoring agents, and the like can be added to the above syrups and drinks.

Parenteral preparations include external preparations such as ointments, creams and solutions, as well as injections. Vaseline, paraffin, oils and fats, lanolin, macrogold and the like are used as the base material for external preparations, and ointments, creams and the like can be prepared by conventional methods.

Injections include liquid formulations and freeze-dried formulations. It is used by aseptically dissolving it in distilled water for injection, physiological saline, or the like.

As a food formulation, it exhibits AGEs adsorption, so it is used in energy drinks and tonic foods. Since it promotes nerve activity, it is used as a food for nerve rehabilitation through genetic disorders in nerve cells and as a meal for learning. It is also used in beauty foods. As a food with health claims, it is preferable to use it as a food with nutrient function claims or a food for specified health use.

When the obtained food preparation is used for pets such as dogs and cats, and livestock animals, it is used as a feed or pet supplement for the purpose of recovering muscle gene disorders, suppressing aging, and improving exercise capacity.

As cosmetics, it can be used together with surfactants, solvents, thickeners, excipients and the like according to conventional methods. For example, it can be in the form of cream, hair gel, face wash, serum, lotion, and the like.

The cosmetic may be in any form, and may be used in the form of solution, cream, paste, gel, gel, solid or powder. Since this derivative dissolves in both water-soluble and oil-soluble solvents, it can be used in a wide range of cosmetics. That is, it can be dissolved in aqueous solutions and oils.

The cosmetics produced here are used for the purpose of repairing damaged genes in the skin, increasing ceramide, collagen and elastin, and maintaining skin health.

In addition, this sphingosine derivative can be used in toothpastes, mouthwashes, toothpastes, etc. for the purpose of maintaining the functions of genetically impaired gingival cells.

In addition, it can be used as a plant activator for the purpose of increasing fruiting and yield by restoring gene disorders in plants.

This plant activator can be used for the purpose of promoting the cultivation of flowers such as high-grade and rare orchids, phalaenopsis and pines, and stabilizes the cultivation of leaves, vegetables and grains. It can also be used for the cultivation of vegetables and leaves in a plant factory, and the cultivation efficiency can be improved.

Next, a method for producing a sphingosine derivative exhibiting an AGEs-adsorbing action, comprising a step of fermenting Laminaria japonica with a marine microorganism, Sphingomonas, and then purifying it with Mitsubishi Chemical's Diaion HP20 will be described.

The sphingosine derivative referred to herein is composed of one molecule of sphingosine represented by the formula (1), pyridine having a carboxylic acid, and three molecules of guluronic acid. All of these bonds are natural, and substances are linked via ester bonds, peptide bonds and ether bonds.

The sphingosine derivatives, sphingosine, carboxylic acid-containing pyridine and guluronic acid, are preferred because they exist in nature, have a lot of dietary experience, and are recognized for their safety.

This derivative also acts on AGEs present in the skin, nerves, bones, muscles, liver, kidneys, etc., adsorbs and excretes AGEs, and restores tissue and bodily functions.

This production method consists of a step of fermenting sea kelp with a marine microorganism, Sphingomonas. Plants and algae containing guluronic acid can also be used instead of Laminaria japonica.

The raw materials are Laminaria japonica and Sphingomonas.

The kelp referred to here is a marine kelp of the genus Laminaria under the scientific name of Saccharina Japonica. It is cultivated in Japan and Asian countries. It has a lot of food experience and its safety has been confirmed.

The edible part of the kelp is used.

The kelp collected in Japan, the United States, Asia, and other countries may be used, but Japanese kelp is preferable because of its high quality and low price.

The Laminaria japonica is preferably dried and powdered, and autoclave sterilization prior to fermentation is preferred for smooth fermentation.

A powder with a particle size of 3 micrometers or less is preferred as it makes the process of fermentation easier to carry out.

The fungus used for fermentation is the marine microorganism Sphingomonas. Sphingomonas is a Gram-negative non-spore-forming aerobic bacillus belonging to the family Sphingomonas, genus Sphingomonas. A representative fungus is Sphingomonas paucimobilis, but any microorganism belonging to the genus Sphingomonas may be used.

A characteristic of Sphingomonas is that it does not have a cell wall but has sphingolipids in the cell membrane like cells. It is a marine microorganism and exists in the waters around Japan. In particular, Sphingomonas from Kyushu is hygienic and excellent in terms of fermentation efficiency.

The amount of each of the above fermentations to be added is preferably 0.001 to 0.03 weight of Sphingomonas per 1 weight of dry powder of Laminaria japonica. It is preferable to pre-culture Bacillus natto before it is fermented, because it shortens the initial fermentation time and shortens the fermentation time.

The fermentation is preferably carried out in clean culture tanks and the ingredients are mixed with sterile tap water.

Also, this fermentation is heated to 38-40° C. and fermented for 2 to 14 days. It is preferable to carry out process control of fermentation by quantifying the target sphingosine derivative by HPLC or TLC and confirming the growth of the cells, because the production amount is adjusted.

The fermentation is preferably carried out in clean culture tanks and the ingredients are mixed with sterile tap water.

A sphingosine derivative produced by this fermentation process is formed.

It is preferable that the fermented product is extracted with water-containing ethanol because the product can be efficiently recovered, the bacteria can be sterilized, and the next step can be easily performed. In addition, it is preferable to subject the obtained fermented product to ultrasonication because the product is easily separated. Concentration by freeze-drying or the like is preferable because the following steps can be performed in a short time. In addition, the purity is increased by combining and repeating the purification process.

It is preferable to separate and purify the desired sphingosine derivative from the above-mentioned reduction reaction product from the viewpoint that the amount of intake can be reduced as a highly pure substance. As a method for this purification, it is purified by Mitsubishi Chemical's Diaion HP20. Further, since the purity increases by repeating this purification, it is preferable to repeat the purification.

The sphingosine derivative of high purity desired is obtained by purifying and fractionating with Mitsubishi Chemical's Diaion HP20. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc., whose surfaces are coated as described below, are used. Those having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the more accurate separation is performed, but there is a disadvantage that the separation time is long.

Furthermore, reversed-phase carriers or resins whose surface is coated with a hydrophobic compound are preferably used for separation of highly hydrophobic substances. Those coated with cationic substances are suitable for separating anionically charged substances. Also, those coated with anionic substances are suitable for separation of cationically charged substances. When coated with a specific antibody, it is used as an affinity carrier or resin that separates only specific substances.

Affinity carriers or resins are used for the specific preparation of antigens using antigen-antibody reaction. Distributable carriers or resins, such as silica gel (manufactured by Merck & Co.), are used to isolate substances when there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorptive carrier or resin, a distributive carrier or resin, a molecular sieve carrier or resin, and an ion-exchange carrier or resin are preferred because they can reduce production costs. In addition, a reversed-phase carrier or resin and a distributive carrier or resin are more preferable because they have a large difference in partition coefficient with respect to the separation solvent.

When an organic solvent is used as the separation solvent, a carrier or resin that is resistant to the organic solvent is used. Also preferred are carriers or resins that are utilized in pharmaceutical or food manufacturing.

From these points, Diaion (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas) as adsorptive carriers, and Sephadex LH-20 (manufactured by Amersham Pharmacia) as a molecular sieve carrier. , silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas Co.) as an ion-exchange carrier, and DM1020T (manufactured by Fuji Silysia Ltd.) as a reversed-phase carrier.

Among these, Diaion HP-20 is preferable because it can purify organic acids, peptides and polyphenols from natural products. Sephadex LH-20 and DM1020T are also more preferred.

The resulting extract is dissolved in a separation carrier or solvent to swell the resin prior to separation. The amount is preferably 2 to 40 times the weight of the extract, more preferably 4 to 20 times the weight of the extract, from the viewpoint of separation efficiency. The separation temperature is preferably 10 to 30°C, more preferably 12 to 25°C, from the viewpoint of the stability of the substance.

As the solvent for separation, water, a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. Methanol, ethanol, propanol, and butanol are used as lower alcohols, and ethanol, which is used as food, is preferred. In addition to water-soluble solvents, it can also be dissolved in oil-soluble solvents such as vegetable oils, fish oils, and animal oils such as lard.

When using Sephadex LH-20, a lower alcohol is preferred as the solvent for separation. When using silica gel, the separation solvent is preferably chloroform, methanol, acetic acid, or a mixture thereof.

When Diaion HP-20 is used, the solvent for separation is preferably a lower alcohol such as methanol or ethanol, or a mixture of a lower alcohol and water.

It is preferable to collect a fraction containing a sphingosine derivative and remove the solvent by drying or vacuum drying to obtain the target sphingosine derivative as a powder or a concentrated liquid, because the influence of the solvent can be excluded.

Moreover, it is preferable to carry out the final extraction with edible oils or fats and oils used in cosmetics, because the obtained sphingosine derivatives are stably maintained. For example, it is preferable to extract with soybean oil, rice bran oil, grapeseed oil, olive oil, and jojoba oil.

Moreover, it is preferable to pulverize this sphingosine derivative for the purpose of preservation.

Hereinafter, the embodiment will be described in detail using examples and test examples. Note that these are only examples, and the conditions can be changed within the scope of common sense according to differences in raw materials and specimens.

Laminaria japonica (scientific name: Laminaria Japonica) cultivated in Hokkaido was purchased from Maruha Nichiro Co., Ltd. and used. After washing the kelp with tap water, it was dried in the sun and pulverized with a grinder (super free mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.0 kg of pulverized kelp. 2 kg of purified water was added thereto, and the mixture was placed in a glass container and autoclaved at 121° C. for 5 minutes.

This was placed in a clean fermentation tank (sterilized round 40-liter tank for fermentation, manufactured by Endo Kagaku), 3 kg of sterilized purified water was added, and the mixture was stirred.

Separately, Sphingomonas (scientific name: Sphingomonas) produced in Nagasaki Prefecture was used as a gift from the Faculty of Fisheries, Nagasaki University. First, 10 g of Sphingomonas was added to 50 mL of sterilized purified water, and 10 g of sterilized soybean powder (produced in Hokkaido) was pre-cultured to prepare a fermentation preparatory liquid. This fermentation preparatory liquid is necessary for the efficient implementation of the following fermentations.

The above precultured Sphingomonas fermentation preparatory liquid and dry powder of Laminaria japonica were added to the fermentation tank, stirred, and then heated in a temperature range of 40 to 44° C. to ferment.

During the fermentation process, the fermented liquid was sampled while performing bubbling and stirring by aeration. It was fermented for 7 days. After completion of fermentation, the fermented product was taken out from the fermentation tank and sterilized by boiling. This fermented product was filtered with a filter cloth and diatomaceous earth to obtain 3.1 kg of a fermented liquid.

The fermentation was sterilized by autoclaving (121° C., 5 minutes) to stop the fermentation. This was filtered again, and the filtrate was used as the target sphingosine derivative. This was designated as Specimen 1.

Furthermore, it was purified using Mitsubishi Chemical's Diaion HP-20. That is, 4 L of purified water containing 7% ethanol was added to 100 g of the sphingosine derivative of the aforementioned specimen 1, and a glass column ( Endo Kagaku).

5 L of 7% ethanol solution was added to this for cleaning, and then 1 L of 40% ethanol solution was added for cleaning. Further, 1 L of 70% ethanol solution was added to elute the target sphingosine derivative, and the eluate was concentrated and purified.

This purification operation was repeated three times. A sphingosine derivative purified by three purification operations was distilled under reduced pressure to remove the ethanol portion, thereby preparing an aqueous solution. This was vacuum-dried to obtain 6.1 g of a purified sphingosine derivative. This sample was designated as Specimen 2. The sphingosine derivative had a purity of 99.5% and a yield of about 6.1%, which was sufficient for production from natural products, confirming that this production method is an excellent production method.

Test methods and results for structural analysis of sphingosine derivatives are described below.
(Test example 1)

Sample 2 obtained as described above was dissolved in deuterated dimethyl sulfoxide (manufactured by Sigma-Aldrich) and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation).

Furthermore, as a result of analyzing this with a nuclear magnetic resonance apparatus (500 MHz, NMR, manufactured by Bruker), as a result of H-NMR measurement in deuterated dimethyl sulfoxide of this purified product, the peak positions are 0.95 and 1.00. , 1.65, 1.66, 1.75, 1.77, 1.79, 1.84, 1.96, 2.08, 2.52, 2.56, 2.75, 2.77, 3 .30, 3.31, 3.32, 3.42, 3.61, 3.67, 3.69, 3.74, 3.91, 3.93, 3.97, 4.02, 4.06 , 4.23, 4.84, 4.86, 5.05, 5.99, 6.02, 6.14, 6.17, 7.55 and 7.58 ppm. These peaks coincided with those of chemically synthesized sphingosine derivative standards.

Further, when this sphingosine derivative was structurally analyzed by C-NMR at 500 MHz in deuterated dimethylsulfoxide, the peak positions were 13.2, 13.5, 14.3, 16.4, 32.4, 32.8, 35.7, 41.4, 41.5, 42.0, 42.9, 44.5, 48.6, 52.4, 63.3, 63.5, 67.0, 67. 7, 72.2, 72.3, 75.4, 75.5, 78.6, 78.7, 79.1, 79.3, 80.6, 95.5, 95.8, 101.3, 101.6, 110.3, 111.5, 124.6, 125.4, 131.7, 133.1, 154.7, 155.9, 169.9, 170.0, 174.5 and 175. 3 ppm was found. These peaks coincided with those of chemically synthesized sphingosine derivative standards.

A chart of the 13C-NMR analysis results is shown below. (The unit of horizontal axis is ppm, and the unit of vertical axis is peak intensity.)

Further, the HPLC analysis of Specimen 2, which is a purified product, showed one main peak, and impurities were less than 0.5%. The purity of the target sphingosine derivative contained in sample 1 was 69%.

From the above analysis results, these chemically synthesized peaks exhibited the same structure as the chemically synthesized sphingosine derivative standard product, and could be identified as the target sphingosine derivative. Further, when the sample 2 was pulverized, as a result of dissolving it in an aqueous solution, generation of hydrogen gas was confirmed by gas chromatography (manufactured by Shimadzu Corporation). The amount of hydrogen gas generated in this case was 1.6 ppm.

A skin effect test using human skin epidermal cells under the condition of adding AGEs is described below. In addition, this test method is a reproducible conventional method that can biochemically verify the effects of the ingredients.
(Test example 2)

Human-derived epidermal cells (epidermal-derived epidermal cells) purchased from Kurabo were used. 1000 cells cultured using 5% fetal bovine serum-containing MEM medium (manufactured by Sigma) as a culture medium were seeded in a 35 mm culture petri dish (manufactured by FALCON) together with 1 mL of culture medium, and cultured at 37° C. under 5% carbon dioxide gas. bottom. 10 mg of human albumin-derived AGEs (manufactured by Funakoshi) was added thereto. Samples 1 and 2 and EGF (manufactured by Funakoshi, epidermal growth factor) as a positive control were all added at a final concentration of 0.1 mg/ml. This was cultured for 48 hours and subjected to the following tests.

After collecting the culture medium, the viability of epidermal cells was counted under a microscope by the trypan blue method. A cell suspension of epidermal cells was then prepared. The amount of AGEs contained in this cell suspension was spectroscopically quantified by an ELISA method (OxiSelect, manufactured by Cosmo Bio). That is, using AGE-modified BSA as a standard substance, color was developed with an anti-AGEs antibody and HRP, and the amount of AGEs was quantified by measuring the absorbance at 450 nm. Five petri dishes were used, and the average value was calculated. It was compared with a solvent control group to which only solvent was added.

As a result, addition of 0.1 mg/ml of Specimen 1 increased the number of human-derived epidermal cells to an average value of 180% compared to the solvent control group. In sample 2, it increased to 242%. On the other hand, EGF showed 168%. As a result, specimens 1 and 2 exhibited better cell proliferation than EGF. Moreover, when the sample 1 and EGF were added simultaneously, the cell count was 409%, confirming the synergistic action of the sample 1 and EGF.

The amount of AGEs in the cell suspension was 339 µg in the solvent control group, 36 µg in the sample 1-treated group, 12 µg in the sample 2-treated group, and 324 µg in the EGF-treated group. As a result, it was confirmed that Samples 1 and 2 act to reduce AGEs.

The amount of 8OHdG in the cells was 677 ng in the solvent control group, 83 ng in the sample 1-treated group, 41 ng in the sample 2-treated group, and 640 ng in the EGF-treated group.

8OHdG is a state in which the gene is modified by active oxygen or AGEs and oxidized, and represents gene disorder. This value was low in specimens 1 and 2, and was superior to the action of EGF. This is due to the anti-oxidation effect of the genes due to the adsorption of AGEs by the specimens 1 and 2.

On the other hand, in a skin irritation experiment using artificial skin EpiSkin (manufactured by SkinEthic) as part of the safety test, no irritation was observed with the addition of Samples 1 and 2, and safety was confirmed. This method has been established as a cell-based skin irritation test evaluation method that does not use animals.

The adsorption of BSA-bound AGEs by specimens 1 and 2 was examined below.
(Test example 3)

1 mg of BSA-bound AGEs purchased from Cosmo Bio was placed in a test tube together with 1 mL of phosphate buffer (PH 7.4). To this was added 1 mL of a 1 mg/mL solution of sample 1 and sample 2. 1 mL of purified water was used as solvent control. In addition, 1 mg of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a control substance.

The test tube containing these was allowed to stand at 37° C. for 1 hour to allow the AGEs and the sample to adsorb. After 1 hour, the reaction solution was sampled and applied to a column filled with an ion exchange resin (Dowex, weakly acidic cation exchange resin type), and the filtrate was collected. It was previously confirmed that the AGEs adsorbed to the specimens 1 and 2 were adsorbed to the ion exchange resin and the free AGEs were excreted.

The amount of AGEs contained in the resulting filtrate was spectroscopically quantified by an ELISA method (OxiSelect, manufactured by Cosmo Bio). The experiment was performed 5 times, and the average value of the obtained data is shown.

As a result, in the solvent control, the amount of AGEs in the filtrate was 0.93 mg. The amount of AGEs in the filtrate with the addition of specimen 1 was 0.13 mg, and the adsorption rate relative to the solvent control was 86%. The amount of AGEs in the filtrate with the addition of sample 2 was 0.04 mg, and the adsorption rate relative to the solvent control was 96%. In the case of activated carbon treatment, the amount of AGEs in the filtrate was 0.92 mg. The adsorption rate was 1.1%.

As a result of the above, it was found that the specimens 1 and 2 exhibited AGEs adsorption and reduced AGEs. On the other hand, no AGEs-adsorbing action was observed with activated carbon.

The sphingosine derivative obtained in the present invention exhibits AGEs-adsorptive action and alleviates diseases and cell damage caused by glycation. This will improve the quality of life of the people, increase the healthy working population, and reduce medical expenses.

The sphingosine derivative obtained by the present invention exhibits an AGEs-adsorbing action, which increases the number of skin cells, and contributes to the improvement of the skin of those suffering from skin troubles such as spots, wrinkles, and sagging due to saccharification. contribute to the development of

Since the sphingosine derivative obtained by the present invention is produced by a fermentation method, it can be used as a functional food and contributes to the development of the food industry and the fermentation industry.

Claims (1)

A sphingosine derivative exhibiting an AGEs-adsorbing action represented by the following formula (1).