JP2017140000A - Neuraminic acid derivative exhibiting langerhans cell-activating action and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a neuraminic acid derivative exhibiting Langerhans cell-activating action and a method for producing the same.SOLUTION: According to the invention, a neuraminic acid derivative of interest is a derivative consisting of one molecule of neuraminic acid and one molecule of glutamine, which are bound by a peptidic linkage. A method for producing such a derivative consists of the steps of adding branched cyclodextrin to a fermentation broth in which Morchella esculenta and soybean are fermented with Bacillus natto. Here, the neuraminic acid derivative can also be used for health foods, cosmetics, medicines, and quasi-drugs for the purpose of immunomodulation, anti-allergy, skin-beautifying action, measures against atopy, and antioxidant action by generation of hydrogen gas. A synergistic action with EGF is also recognized.SELECTED DRAWING: None

Description

The present invention relates to a neuraminic acid derivative exhibiting a Langerhans cell activation action and a method for producing the same.

Langerhans cells are immune cells that control the immune function of the skin. It is also highly linked to the nervous system, and has the function of transmitting cranial nerve changes to the skin.

Furthermore, Langerhans cells move inside the epidermis cells of the skin and monitor the entry of foreign substances such as bacteria and viruses. When bacteria and viruses invade the skin, Langerhans cells increase dendrites, detect foreign substances through the receptors at their tips, and release immune cytokines.

The Langerhans cells decrease with age, and the barrier function of the skin decreases with age. Activating Langerhans cells is important for recognizing foreign bodies and protecting the skin.

In addition, Langerhans cells sense UV and radiation and protect the skin from these stimuli. The invention relating to Langerhans cells includes a whitening method and a screening method for a melanin synthesis inhibitor. Here, β-glucan stimulates Langerhans cells to suppress melanin production (see, for example, Patent Document 1).

In addition, there is an invention of activation of immune cells including Langerhans cells. In this document, an adjuvant for transcutaneous immunization activates Langerhans cells (see, for example, Patent Document 2). However, here, the activation of Langerhans cells is mild and its industrial use is limited.

There is no specific example of a structure that activates Langerhans cells, and the technique used is described, but this is not a method for increasing and activating Langerhans cells themselves.

However, there is no invention relating to a method for increasing Langerhans cells in the medical area, skin area or cosmetic area, an active ingredient thereof, or a useful substance.

Thus, when the substance that increases Langerhans cells is a chemical substance, there is a risk of causing side effects, and industrial use is limited.

Therefore, a substance derived from a natural product and activating Langerhans cells is desired.

JP2014-80386 JP 2010-59201 A

As described above, the Langerhans cell activation action by existing natural products is mild, and there is a problem that the industrial use is limited, and there is a problem in safety with chemically synthesized substances. limited.

Therefore, a natural product exhibiting an excellent Langerhans cell activation action with weak side effects and a production method for efficiently producing the natural product are desired.

In order to achieve the above object, the invention described in claim 1 relates to a neuraminic acid derivative exhibiting a Langerhans cell activating action represented by the following formula (1).

In order to achieve the above object, the invention described in claim 2 relates to a method for producing a neuraminic acid derivative exhibiting a Langerhans cell activation action.

Since this invention is comprised as mentioned above, there exist the following effects.

According to the derivative of claim 1, it is possible to exert an excellent Langerhans cell activation effect.

According to the manufacturing method of Claim 2, the neuraminic acid derivative which exhibits a Langerhans cell activation effect | action efficiently can be manufactured.

Hereinafter, embodiments embodying the present invention will be described in detail.

First, the neuraminic acid derivative exhibiting the Langerhans cell activation action represented by the following formula (1) is composed of one molecule of neuraminic acid and one molecule of glutamine.

Neuraminic acid in this derivative is a kind of carbohydrate, exists on the cell membrane of various cells, and is involved in cell functions. It consists of 9 carbons, 17 hydrogens, 1 nitrogen and 8 oxygens.

Neuraminic acid is often present as a derivative. For example, sialic acid and N-acetylneuraminic acid are present as substitutes at the N or O position.

In addition, there are derivatives in which an acetyl group, a lactic acid group, a methyl group, and a phosphoric acid group are bonded. These derivatives differ in function and are related to cell differentiation and function. Some of them function as receptors.

In this neuraminic acid derivative, the amino group of glutamine is bonded to the carboxylic acid moiety of neuraminic acid. Glutamine is L-type and is a type constituting peptides and proteins. This bond is a peptide bond, and the carbon of the carboxyl group of neuraminic acid and the N group of glutamine are directly bonded.

This neuraminic acid derivative is chemically synthesized and used as a standard product. Its structure is identified by analysis such as H-NMR. For example, as a result of 400 MHz H-NMR analysis, 1.71, 1.89, 1.99, 2.14, 2.46, 3.18, 3.49, 3.51, 3. Detected around 61, 3.74, 3.84 and 8.08 ppm. Furthermore, this neuraminic acid derivative is identified by mass spectrometry, IR, qualitative reaction, etc. of a standard product.

Other hydroxyl groups are free types and retain reactivity as hydroxyl groups. These hydroxyl groups have an antioxidant action and stabilize this neuraminic acid derivative. Furthermore, it increases water solubility and enhances absorption from the intestinal tract and skin.

The glutamine portion of this neuraminic acid derivative has a slight hydrophobicity and is preferred because penetration into the fat portion is promoted.

This neuraminic acid derivative is preferably amphiphilic due to the water-soluble part of the hydroxyl group and the hydrophobic part of glutamic acid, and is excellent in penetration into the stratum corneum of the skin from the viewpoint of the effect. Since Langerhans cells are present in the epidermis layer, which is the lower layer of the stratum corneum, it is preferable that this neuraminic acid derivative can directly act on Langerhans cells.

The neuraminic acid derivative penetrates into the mucous membrane of the small intestine, is easily absorbed from the intestinal tract, and is preferably transported to the skin of the whole body via the liver.

This neuraminic acid derivative penetrates the epidermal tissue and activates Langerhans cells. Langerhans cells have a function of regulating immunity, and are preferable because they exhibit both the functions of activating immunity against a reduced immune function and suppressing the immune function that is too high. In particular, it is excellent in enhancing the immune function by activating Langerhans cells in a state of reduced immunity, and it is preferable to increase the immune function reduced by aging, irradiation, sunburn or damage caused by ultraviolet rays.

This neuraminic acid derivative stimulates various receptors on the cell surface of Langerhans cells to activate the cells, and regulates the antigen presentation ability.

Furthermore, it is preferable to act synergistically with growth factors such as EGF and SCF. That is, EGF binds to the EGF receptor of skin epithelial cells and activates the intracellular signal transduction system, but this neuraminic acid derivative increases the reactivity of the EGF receptor and stimulates the intracellular signal transduction system. This activates the function of EGF.

In addition, neuraminic acid derivatives pass through the cell membrane of Langerhans cells and are taken into cells, and act on immune regulatory genes, growth factor genes, cytokine genes, etc. in the nucleus to activate Langerhans cells. Furthermore, it is preferable that neuraminic acid derivatives activate Langerhans cells against skin cancer, increase immune function, particularly natural killer activity, increase phagocytosis of skin cancer, and reduce cancer.

When this neuraminic acid derivative is powdered, it reacts with a water-soluble solvent in a reduced state to generate hydrogen gas. This is a reaction by the hydroxyl group of neuramin, and the generated hydrogen gas is industrially advantageous in that it can eliminate the influence of active oxygen by erasing the hydroxyl radical, and is preferably used for cosmetics and foods.

On the other hand, this neuraminic acid derivative is decomposed by esterase in the kidney to be decomposed into neuraminic acid and glutamine, metabolized and excreted, so it is highly safe and has low persistence in the environment such as the body and soil. This is preferable from the environmental aspect.

This neuraminic acid derivative is also produced by any method such as a fermentation method, an enzyme reaction method or a chemical synthesis method.

For example, the neuraminic acid derivative can be extracted from morel mushrooms. As this extraction method, a method using fermentation or a digestive enzyme such as protease or lipase is preferable because the extraction efficiency is improved.

Moreover, in the case of an enzyme reaction method, it can extract from the plant containing neuraminic acid. Alternatively, it can be biosynthesized by microorganisms by fermentation.

Furthermore, as a purification method, it is preferable to use a purification operation such as a separation resin.

For example, it is preferably separated and separated by a separation carrier or resin. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc., whose surfaces are coated as described later, are used. Those having a particle size of 0.1 to 300 μm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time.

For example, a reverse phase carrier or resin whose surface is coated with a hydrophobic compound is used for separation of a highly hydrophobic substance. Those coated with a cationic substance are suitable for the separation of anionically charged substances. Also, those coated with an anionic substance are suitable for separating a cationically charged substance. When a specific antibody is coated, it is used as an affinity carrier or resin for separating only a specific substance.

The affinity carrier or resin is used for specific preparation of an antigen using an antigen-antibody reaction. A partitionable carrier or resin is used for isolation of a substance such as silica gel (manufactured by Merck) if there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing production costs. Furthermore, the reverse phase carrier or resin and the dispersible carrier or resin are more preferable because the difference in the distribution coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Moreover, the carrier or resin used for pharmaceutical manufacture or food manufacture is preferable. From these points, Diaion (Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH-20 (Amersham Pharmacia) is used as the molecular sieve carrier. Silica gel as the distribution carrier, IRA-410 (Rohm and Haas) as the ion exchange carrier, and DM1020T (Fuji Silysia) as the reverse phase carrier are more preferable.

Of these, Diaion, Sephadex LH-20 and DM1020T are more preferred.

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. The amount thereof is preferably 1 to 40 times the weight of the extract from the viewpoint of separation efficiency, and more preferably 4 to 20 times. The separation temperature is preferably 4 to 30 ° C., more preferably 10 to 25 ° C. from the viewpoint of the stability of the substance.

As the separation solvent, water or a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, and ethanol used for food is preferable.

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

When Diaion and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water.

In addition, it is preferable to collect a fraction containing the activity and remove the solvent by drying or vacuum drying to obtain a powder or a concentrated liquid because the influence of the solvent can be excluded.

This neuraminic acid derivative acts directly on Langerhans cells and exhibits skin beautifying and skin regeneration.

It is preferable to add fats and oils to the neuraminic acid derivative because the resulting active ingredient is stably maintained in the oil. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

It is used as an injectable or parenteral agent such as an oral agent or a coating agent as a pharmaceutical, and as a quasi-drug, it is used in a tablet, capsule, drink, soap, coating agent, gel, toothpaste, etc. The

Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, excipient, swelling agent, lubricant, sweetener, flavoring agent and the like. The tablets can also be coated with shellac or sugar.

Moreover, in the case of the said capsule, liquid carriers, such as fats and oils, can be further contained in said material. In the case of the above syrup and drink, sweeteners, preservatives, pigment flavoring agents and the like can be added.

Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams, and liquids. Vaseline, paraffin, fats and oils, lanolin, macro gold, etc. are used as a base material for external preparations, and can be made into ointments, creams, and the like by ordinary methods.

Injections include liquids, and other lyophilization agents. This is used aseptically dissolved in distilled water for injection or physiological saline at the time of use.

It is used as a food preparation for beauty foods intended for beauty, foods intended for beauty, immune function activators intended to maintain healthy cells, and the like. Moreover, it is preferable to use as a health functional food for a nutritional functional food or a food for specified health use.

When the obtained food preparation is used for pets and livestock animals such as dogs and cats, it is used as a feed or a supplement for pets for the purpose of maintaining the health of the skin and tissues.

As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of cream, gel for hair, facial cleanser, cosmetic liquid, lotion, etc., and becomes a cosmetic that promotes the production of Langerhans cells.

The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder.

Next, the manufacturing method of the neuraminic acid derivative which exhibits the Langerhans cell activation effect | action consisting of the process which adds morel mushroom and soybean, and adds a branched cyclodextrin to the fermented liquid fermented with natto and performs protease treatment is demonstrated.

The neuraminic acid derivative here is a substance composed of one molecule of neuraminic acid and one molecule of glutamine, and is a substance exhibiting a Langerhans cell activation action.

This neuraminic acid derivative penetrates the epidermal tissue and activates Langerhans cells. Langerhans cells have a function of regulating immunity, and are preferable because they exhibit both the functions of activating immunity against a reduced immune function and suppressing the immune function that is too high.

In particular, it is excellent in enhancing the immune function by activating Langerhans cells in a state of reduced immunity, and it is preferable to increase the immune function reduced by aging, irradiation, sunburn or damage caused by ultraviolet rays.

This neuraminic acid derivative activates cells by stimulating various receptors on the cell surface of Langerhans cells, and regulates the antigen presentation ability.

This production method comprises a step of adding protease and soybean, adding a branched cyclodextrin to a fermentation broth fermented with Bacillus natto, and subjecting it to protease treatment.

The starting materials are morel mushroom, soybean, natto, branched cyclodextrin and protease.

The morel mushroom has the scientific name Morchella esculenta, and is an edible mushroom belonging to the genus Amigasatake.

Agaric can be used either naturally occurring or cultivated from spores. In the case of the indigenous nature, it may be any of the northern hemispheres such as Japan, Asia, Europe, and the United States.

When cultivated from spores, it can be grown using potato and glucose agar medium or malt extract agar medium.

By spore seeding on these media and culturing at 20 ° C. within 24 hours, germination and hyphae are obtained.

This country of origin can be used in Japan, Asia, America, Europe, etc. In particular, Japanese mushrooms are preferred because of their good quality.

The soybean used as a raw material can be any soybean from Japan, China, the United States, Russia, etc., but it is preferable that the soybean is traceable and the producer is clear.

Of these, soybeans cultivated organically or without agricultural chemicals are more preferred because they do not contain harmful agricultural chemicals or metals.

When using soybeans, Nara Machinery Co., Ltd. free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko as vacuum dryer, small size manufactured by Matsui Co., Ltd. It is pulverized by a pulverizer such as a vacuum heat transfer dryer DPTH-40, clean dry VD-7, VD-20 manufactured by AKM Kyushu Technos Co., Ltd., DM-6 manufactured by Nakayama Technical Research Institute. Thereby, the process of fermentation tends to advance efficiently.

The Bacillus natto used is the scientific name Bacillus subtilis, which is a useful microorganism used in the production of natto. Natto bacteria, a powder of Natto Motopo, is preferable because of its good quality and suitable for fermentation.

A branched cyclodextrin is one of cyclic glucose, and is preferable because glucose is bound cyclically and used in foods and cosmetics. Since this branched cyclodextrin has a hydrophobic portion in the lumen, it is easy to adsorb highly hydrophobic substances. Branched cyclodextrins manufactured by Shimizu Minato Sugar Co., Ltd. are preferred because of their high quality.

As the protease to be used, the quality of protease A “Amano” SD, protease M “Amano” SD or protease P “Amano” 3SD, which are food processing proteases manufactured by Amano Enzyme, is preferable because of its stable use.

First, the mushroom is pulverized by a pulverizer or a mixer. By being pulverized, it becomes easy to process. The soybeans are pulverized by a pulverizer and suspended by adding clean water. 60-200g of soybeans are added to 100g of mushroom, and they are stirred together with 3 liters to 10 liters of water such as purified water in a clean container.

Morel and soybeans are sterilized by boiling and added to the fermentation tank. By sterilization, contamination with various bacteria is prevented, and fermentation with Bacillus natto proceeds.

Fermentation may be either a stationary method or a stirring method, but a stirring method is preferred because fermentation can be performed in a short time. Fermentation is preferably carried out at 40 to 44 ° C. for 21 to 74 hours. When the temperature is low and the time is short, the fermentation does not proceed, and when the temperature is high and the time is long, the target neuraminic acid derivative may be decomposed.

The fermentation broth is preferably filtered with a filter cloth or the like because the following steps can be easily performed.

Branched cyclodextrin is added to the filtrate. A branched cyclodextrin is one of cyclic glucose, and is preferable because glucose is bound cyclically and used in foods and cosmetics. Since this branched cyclodextrin has a hydrophobic portion in the lumen, it is easy to adsorb highly hydrophobic substances. Branched cyclodextrins manufactured by Shimizu Minato Sugar Co., Ltd. are preferred because of their high quality.

The branched cyclodextrin added is preferably 30 to 300 g of branched cyclodextrin per 100 g of mushroom. This branched cyclodextrin binds neuraminic acid and glutamine to produce a derivative.

The suspension with the branched cyclodextrin is preferably stirred.

Protease is added to this suspension. The protease to be added is preferably 0.003 to 0.5 g with respect to 100 g of the fermentation broth. It is preferable that the protease is suspended in purified water because the reaction proceeds.

This suspension is preferably warmed and stirred to promote the reaction. As heating, 37-44 degreeC is preferable. The stirring is preferably 13 to 37 times per minute. The time is preferably 1 to 5 hours.

This protease reaction solution is filtered. Efficient filtration is achieved by using filter paper or membrane filter. It is preferable because unreacted components and raw materials can be excluded by filtering to obtain a filtrate.

The obtained reaction product is sterilized by boiling to inactivate the protease.

The obtained reaction product is pulverized by lyophilization and used.

Separating and purifying the desired neuraminic acid derivative from the reaction product is preferable from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin.

For example, the desired neuraminic acid derivative is obtained by separation with a separation carrier or resin and fractionation. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc., whose surfaces are coated as described later, are used. Those having a particle size of 0.1 to 300 μm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time.

For example, a reverse phase carrier or resin whose surface is coated with a hydrophobic compound is used for separation of a highly hydrophobic substance. Those coated with a cationic substance are suitable for the separation of anionically charged substances. Also, those coated with an anionic substance are suitable for separating a cationically charged substance. When a specific antibody is coated, it is used as an affinity carrier or resin for separating only a specific substance.

The affinity carrier or resin is used for specific preparation of an antigen using an antigen-antibody reaction. A partitionable carrier or resin is used for isolation of a substance such as silica gel (manufactured by Merck) if there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing production costs. Furthermore, the reverse phase carrier or resin and the dispersible carrier or resin are more preferable because the difference in the distribution coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Moreover, the carrier or resin used for pharmaceutical manufacture or food manufacture is preferable.

From these points, Diaion (Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH-20 (Amersham Pharmacia) is used as the molecular sieve carrier. Silica gel as the distribution carrier, IRA-410 (Rohm and Haas) as the ion exchange carrier, and DM1020T (Fuji Silysia) as the reverse phase carrier are more preferable.

Of these, Diaion, Sephadex LH-20 and DM1020T are more preferred.

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. The amount is preferably from 1 to 32 times, more preferably from 5 to 19 times the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 4 to 30 ° C., more preferably 10 to 26 ° C. from the viewpoint of the stability of the substance.

As the separation solvent, water or a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, and ethanol used for food is preferable.

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

When Diaion and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water.

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

Further, it is preferable to powder this neuraminic acid derivative for the purpose of preserving.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. These are merely examples, and conditions can be changed within the range of common sense according to differences in materials, raw materials, and specimens.

1 kg of French mushroom bamboo was purchased from Paseo Co., Ltd. and used. This was washed with water and then suspended with purified water in a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 0.9 kg of a suspension.

Morel pesticides were analyzed in advance for pesticides and confirmed that there were no resident pesticides. It was stored frozen until use.

Furthermore, 1 kg of Hokkaido soybean was purchased from Miwa Bussan and used. This was washed and then pulverized by a pulverizer to obtain about 950 g of a pulverized product.

500 g of suspension of morels and 500 g of soybean powder were transferred to a clean stainless steel cylinder, and 5 liters of purified water was added and suspended.

This was sterilized by boiling at 93-97 ° C. for 1 hour. These were transferred to a stirred fermentation tank (FP211) manufactured by Yokogawa Electric Corporation with a capacity of 100 kg, and 20 liters of sterilized purified water was added.

To this, 3 g of powdered Bacillus natto manufactured by Takahashi Yuzo Laboratory Co., Ltd., the natto bacillus main office, was purchased. This Bacillus natto was suspended in 100 g of sterilized water, powdered soybean powder was added thereto, and the mixture was heated at 37 ° C. for 1 hour and precultured.

This natto fungus solution was added to the agitated fermentation tank and fermented at 41-43 ° C. for 36 hours. The state of fermentation was monitored by degradability of soybean powder and quantification of dissolved protein (Burelet method).

After fermentation, the supernatant of the obtained fermentation broth was roughly filtered through a filter cloth to obtain a filtrate.

22 liters of this filtrate was transferred to a clean tank, and 200 g of branched cyclodextrin (Iso Elite) manufactured by Shisui Minato Seika Co., Ltd. was added thereto and stirred sufficiently.

Furthermore, 3 g of protease M “Amano” SD manufactured by Amano Enzyme was added, and the mixture was heated to 37 ° C. and stirred.

Stirring was performed for 3 hours at room temperature using a stirrer. The reaction solution was sterilized by boiling to deactivate the enzyme. The obtained reaction liquid was subjected to suction filtration with Toyo filter paper (No. 2) to obtain a filtrate.

This solution was freeze-dried with a freeze dryer (freeze trap VA-140S manufactured by Taitec Co., Ltd.) to obtain 136 g of the desired powder. This was designated as Sample 1. When 0.1 g of Sample 1 was suspended in 10 mL of purified water, it was detected by gas chromatography that 1.6 ppm of hydrogen gas was generated.

100 g of the obtained powder of the specimen 1 was suspended in 400 mL of purified water and subjected to 500 g of Diaion (manufactured by Mitsubishi Chemical) swollen with 5% ethanol. After washing with 900 mL of 6% ethanol, it was further washed with 1 liter of 50% ethanol.

To this, 700 mL of 80% ethanol was added to fractionate the desired neuraminic acid derivative. The obtained fraction was dried with a vacuum dryer (manufactured by Nippon Biocon) to obtain 13 g of powder. This powder was designated as Sample 2. When 0.1 g of Sample 2 was suspended in 5 mL of purified water, it was detected by gas chromatography that 1.6 ppm of hydrogen gas was generated.

Below, the test method regarding the structural analysis of a neuraminic acid derivative and a result are demonstrated.
(Test Example 1)

The specimen 2 obtained as described above was dissolved in purified water, filtered, and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation).

Furthermore, it analyzed with the nuclear magnetic resonance apparatus (NMR, the product made from Bruker). As a result of structural analysis, a derivative in which neuraminic acid and glutamine were bound was detected from specimen 2.

The bond was a peptide bond in which one molecule each of the carboxyl group of neuraminic acid and the amino group of glutamine were bonded. On the other hand, the hydroxyl group of inoramic acid was free. Other bonds and polymers were not observed.

As a result of analysis by H-NMR (400 MHz, manufactured by Bruker), chemical shifts were 1.71, 1.89, 1.99, 2.14, 2.46, 3.18, 3.49, 3.51, 3 Peaks were observed at .61, 3.74, 3.84 and 8.08 ppm. Furthermore, neuraminic acid derivatives were identified from the results of IR and mass analysis.

Below, the confirmation test of the effect | action with respect to a human skin origin Langerhans cell is described.
(Test Example 2)

After obtaining an informed concept under the guidance of a doctor from three female volunteers aged 25 to 46 years, skin was collected by an exfoliation method under anesthesia. This was washed with a disinfectant solution and then dispersed in a collagenase-containing phosphate buffer to collect a cell suspension.

The above cell suspension was added to a petri dish (manufactured by Falcon) to which an anti-Langerhans cell antibody was adhered, and allowed to react at room temperature for 1 hour under aseptic conditions.

As the antibody, human-derived Langerhans cell antibody 4D12 mouse IgG purchased from Cosmo Bio was used.

This method is a method of collecting cells by the antigen-antibody method, and the cell recovery rate is as efficient as about 90%.

After the antigen-antibody reaction, the petri dish was carefully washed with a culture solution (5% human serum-containing MEM medium) and cultured in a culture solution at 37 ° C. under 5% carbon dioxide gas.

Cultivation was carried out for 24 hours, and after completion of the cultivation, it was further gently washed with a culture solution. Cells attached to the petri dish were collected with trypsin and used as Langerhans cells.

This was suspended in a culture solution and seeded on a petri dish. This was cultured, and after 18 hours, 0.1 mg / mL of the culture fluid suspension of Specimen 2 was added. Only the culture medium was used as a solvent control. As a growth factor control, 0.1 mg / mL human-derived EGF was added. Furthermore, the group which adds EGF and the sample 2 simultaneously was also set.

This was cultured at 37 ° C. for 48 hours, and the number of viable cells was counted by trypan blue dye method.

Furthermore, the amount of ATP produced in the cell suspension was quantified by a fluorescence method (CellTiter-Glo assay, Promega). In addition, it experimented with 5 petri dishes and the result calculated | required the average value by the change rate with respect to a solvent control.

As a result, the number of Langerhans cells was increased to 320% by adding Sample 2 with the cell number of the solvent control as 100%. On the other hand, EGF is 180%. Sample 2 was superior. Further, the combined use of EGF and Sample 2 resulted in a cell number of 720%, and a synergistic proliferation effect was observed.

As for the amount of ATP, when the value of the solvent control was 100%, the amount of ATP increased to 530% by the addition of Sample 2. On the other hand, EGF was 240%, and the specimen 2 was superior in activation due to increased ATP production in Langerhans cells. Furthermore, the combined use of EGF and Sample 2 resulted in an ATP amount of 1530%, and a synergistic increase effect was observed.

The neuraminic acid derivative obtained in the present invention exhibits a Langerhans cell activating action, and can generate hydrogen gas to remove active oxygen and has few side effects. Can improve the QOL of the people.

Since the method for producing a neuraminic acid derivative obtained in the present invention can also be used as food, it contributes to the development of the food industry.

The neuraminic acid derivative obtained in the present invention has a Langerhans cell activating action and an antioxidant action by hydrogen gas, so it is also used as a cosmetic for skin improvement and contributes to the development of the cosmetic industry.

Claims (2)

A neuraminic acid derivative exhibiting a Langerhans cell activation action represented by the following formula (1).

A method for producing a neuraminic acid derivative exhibiting a Langerhans cell activation effect comprising a step of adding a branched-chain dextrin to a fermented liquid fermented with natto and fermented with Bacillus natto and fermented with natto.