JP2018193339A - Peptide glycan derivative exhibiting anti-inflammatory activity - Google Patents

Abstract

To provide a peptide glycan derivative that exhibits anti-inflammatory activity.SOLUTION: A target peptide glycan derivative is a derivative that consists of phenyl group, ribose, cysteine and arginine. The derivative exerts anti-inflammatory activity to increase keratin in a skin cell. Further, the derivative generates hydrogen gas. A method for producing the derivative consists of a process of adding branched cyclodextrin to fermentation liquid formed by fermenting a swallow nest by Bacillus natto and performing protease treatment to the liquid. The peptide glycan derivative can be used for health food, cosmetic, medicinal drug, quasi drug or plant activator.SELECTED DRAWING: None

Description

The present invention relates to peptidoglycan derivatives exhibiting anti-inflammatory action.

Inflammation is a biological reaction that occurs systemically or locally, and exhibits symptoms such as pain, itching, and redness. An inflammatory reaction occurs when a living body is stimulated by infection with a virus, bacteria, foreign matter, or abrasion.

During inflammation, biological substances such as inflammatory cytokines, inflammatory substances and inflammatory enzymes are produced, causing an inflammatory reaction. Inflammation often becomes an obstacle in daily life, such as fever and headache due to viral infection. In the skin, contact allergies and atopy also have an inflammatory reaction, impairing skin health.

Therefore, substances exhibiting anti-inflammatory action have been developed. Synthetic steroids suppress inflammation, but opportunistic infection is observed as a side effect. In addition, nonsteroidal anti-inflammatory agents called NAIDs also have side effects, such as intestinal abnormalities and vomiting. Therefore, a substance that exhibits an anti-inflammatory action derived from a natural product having no side effect is being searched.

Although there is an invention relating to a composition containing amidine and alkane polyol as an invention relating to a natural anti-inflammatory agent, a substance that exerts an anti-inflammatory effect has not been specified more specifically. (For example, refer to Patent Document 1.)

Moreover, although there exists invention of useful polypeptide as invention regarding the anti-inflammatory effect by a peptide, the effect | action is unclear and the useful substance has not been specified. (For example, see Patent Document 2.)

Thus, although natural products that exhibit anti-inflammatory effects and have few side effects are desired, there is no invention relating to useful components and substances.

Therefore, a substance or a derivative derived from a natural product and exhibiting an anti-inflammatory action is desired.

Japanese Patent No. 4217740 Japanese Patent Application No. 2010-004295

As described above, the anti-inflammatory effect of 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, and the use is limited. ing.

Therefore, a substance exhibiting an excellent anti-inflammatory action with a weak side effect is desired.

In order to achieve the above object, the invention described in claim 1 relates to a peptidoglycan derivative exhibiting an anti-inflammatory action represented by the following formula (1).

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

According to the derivative of claim 1, it is possible to exhibit an excellent anti-inflammatory effect.

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

First, the peptidoglycan derivative exhibiting an anti-inflammatory action represented by the following formula (1) is composed of 27 carbon elements, 39 hydrogen elements, 13 oxygen elements, 5 nitrogen elements, and 1 sulfur element.

That is, the chemical formula of C27H39O13N5S1. It consists of a dipeptide consisting of L-cysteine and L-arginine, one molecule of phenoxy group and two molecules of D-ribose.

The phenyl group portion of this derivative is ester-bonded to the hydroxyl group of ribose. Ribose has an ether bond. The phenyl moiety of this derivative reduces inflammatory prostaglandins such as prostaglandin E2 by inhibiting the activity of COX-2 (cyclooxygenase type 2) induced by inflammation. This decrease in prostaglandin E2 is due to antagonistic inhibition of COX-2. On the other hand, there is a selectivity that does not affect COX-1.

The cyclic part composed of the peptide of this derivative and ribose suppresses the production of inflammatory cytokines. Inflammatory cytokines include TNF alpha, a derivative that suppresses the production of TNF alpha. This suppression occurs when this derivative suppresses the expression of TNF alpha mRNA.

This derivative exerts an anti-inflammatory effect by suppressing inflammatory prostaglandins and inflammatory cytokines. In addition, this derivative has a function of suppressing the production of bradykinin and suppressing the production of inflammatory protease.

The constituent ribose is D-type and natural type. Although ribose is a component of nucleic acids, its physiological action is diverse. This ribose is also used as a fermented food and cosmetics, and its safety has been confirmed.

Cysteine and arginine constituting the peptide are natural L-forms. The safety of these amino acids has also been confirmed. Cysteine has an SH group and exhibits a reduced state. Arginine is a supply source of nitric oxide, and nitric oxide acts on the vascular endothelium to increase blood flow and exerts a relaxing action.

This peptidoglycan derivative can be synthesized organically using phenyl group, cysteine, arginine and ribose as raw materials. This organically synthesized derivative is used for analysis and analysis as a standard substance. However, it is costly to manufacture and uses an organic solvent, so that there are safety disadvantages that it is difficult to use in the cosmetics and food fields.

About the structure of this peptidoglycan derivative, the peak position is 0.92, 1.73, 1.84, and 600 MHz H-NMR (1H-NMR) analysis (made by Bruker) in deuterated dimethyl sulfoxide of this derivative. 2.76, 3.14, 3.23, 3.35, 3.36, 3.43, 3.44, 3.49, 3.65, 3.67, 3.83, 3.88, 3. 94, 4.82, 4.92, 5.03, 5.09, 7.11 and 7.33 ppm.

Further, in the C-NMR (13C-NMR) analysis of this derivative in deuterated dimethyl sulfoxide, the peak positions are 24.2, 25.1, 25.4, 45.7, 50.2, 62.8, 63.1, 68.2, 72.0, 72.1, 75.6, 75.7, 78.7, 78.8, 80.3, 81.6, 81.9, 95.7, 103. 2, 118.9, 132.1, 132.5, 160.7, 173.1 and 173.8 ppm.

Since this peptidoglycan derivative is naturally derived, it has high absorbability and safety. In particular, from the viewpoint of safety, this peptidoglycan derivative is degraded by an internal enzyme, particularly peptidase or esterase. Even if a large amount of this derivative is ingested, the excess amount is decomposed in the living body, so that the safety is high.

This peptidoglycan derivative exhibits an anti-inflammatory effect. In particular, it works to suppress skin inflammation. The reason is degradation by stomach acid in the stomach. That is, it is decomposed by the strong acidity of stomach acid. Therefore, when ingested orally, it is necessary to take measures against gastric acid such as preparation of an acid-resistant preparation or filling into an enteric capsule.

Further, when this derivative is powdered, it generates hydrogen gas when it reacts with an aqueous solution. Since the generated hydrogen gas has a function of removing active oxygen, it is preferable because the active oxygen generated by ultraviolet rays or an oxidizing substance can be removed to stably maintain the living body. Hydrogen gas is preferable because it eliminates hydroxyl radicals, exhibits a reducing action, and exhibits an antioxidant action.

Furthermore, cysteine is a raw material for increasing keratin, and this peptidoglycan derivative has an action of increasing keratin. This peptidoglycan derivative activates keratin synthase to increase the amount of keratin. This increasing action of keratin is preferable because it strengthens the skin and hair.

As a method for extracting or producing this peptidoglycan derivative, there are a fermentation method, an enzyme reaction method, a chemical synthesis method, and the like.

The peptidoglycan derivative can be extracted from beans such as swallow's nest and soybean. In this extraction method, it is preferable to use digestive enzymes such as protease and lipase because the extraction efficiency is enhanced.

In particular, the swallow's nest is preferable as a raw material because it is originally rich in peptidoglycan. Moreover, in the case of an enzyme reaction method, it can extract from the plant containing a peptidoglycan. Alternatively, it can be biosynthesized by microorganisms by fermentation.

Fermenting swallow's nest is useful as a method for producing this peptidoglycan derivative. In the case of fermenting, fermentation technology fermented with natto bacteria together with soybean is preferable because it has abundant knowledge in Japan, has a large track record as an edible product, and is highly safe.

Furthermore, it is preferable to purify for the purpose of obtaining a highly pure derivative. 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 peptidoglycan, silicon oxide compound, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymer and the like whose surface is 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 (manufactured by Mitsubishi Chemical Corporation, HP-20 and HP-21) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH is used as the carrier for the molecular sieve. More preferred is -20 (manufactured by Amersham Pharmacia), silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier.

Of these, Diaion HP-20, 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 HP-20 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 peptidoglycan derivative exhibits an excellent anti-inflammatory action, and is particularly preferable because it can protect against inflammation against external stimuli on the skin. For example, it acts on inflammation caused by bacterial infection, inflammation caused by acne and atopic inflammation. Furthermore, it also biosynthesizes skin keratin and exhibits skin beautifying, anti-inflammatory and skin regenerating effects. It can also be used as an eyelash proliferator, a hair restorer, or a hair cosmetic that requires keratin production.

It is preferable to add fats and oils to the peptidoglycan derivative because the obtained active moiety 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. This derivative is soluble in both water-soluble and oil-soluble solvents. This amphipathic property is preferable because it extends the range of use of this derivative in cosmetics, foods and daily necessities.

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 anti-inflammatory effects, foods intended for beauty and anti-inflammatory effects, and cosmetics for protecting skin by increasing keratin. 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 it becomes a cosmetic that promotes anti-inflammatory and keratin production. The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder. This derivative is soluble in both water-soluble and oil-soluble solvents. This amphiphilic nature is preferred because it broadens the use of this derivative.

It is also used as a plant activator utilizing anti-inflammatory action. In other words, plants cause an inflammatory reaction locally against external enemies such as viruses and bacteria, and their growth is suppressed. Therefore, it is preferable that this derivative activates the growth of the plant by suppressing the inflammation of the plant, resulting in an increase in flowering, fruiting and yield.

In the following, the production of this derivative will be explained by the production process of fermenting swallow's nest. It consists of a step of adding a branched cyclodextrin to a fermented solution obtained by fermenting a swallow's nest with soybeans and natto bacteria and subjecting it to a protease treatment.

The raw materials are swallow's nest, soybean, natto, branched cyclodextrin and protease.

A swallow's nest is a swallow's nest also used for food, for example, a nest of a swallow. Giant swallows are swallows that live on the coast of Southeast Asia. The swallow's nest is thought to be the swallow's saliva solidified, but some of its components are seaweed. Nutritional components include proteins, sugars, lipids, etc., and are used as general food ingredients, particularly as Chinese and Chinese dishes.

The swallow nest to be used is preferably an edible one that is available in Japan because of its high safety. Southeast Asian countries such as Indonesia, Thailand, Philippines, and Malaysia are preferable as production areas, and those that have been imported into Japan and cleaned and disinfected in Japan are more preferable in terms of hygiene.

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 these raw materials, Nara Machinery Co., Ltd. free mill, super free mill, sample mill, goblin, super clean mill, micros, vacuum dryer, Toyo Riko's small vacuum dryer, Matsui Co., Ltd. It is dried and pulverized by a pulverizer such as a small 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 since it has a stable use record.

The swallow's nest is pulverized by a pulverizer and suspended by adding clean water. A 100g swallow's nest is stirred with 1 liter to 10 liters of water such as purified water in a clean container.

The swallow's nest and ground 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 performed at 39 to 44 ° C. for 24 to 72 hours. When the temperature is low and the time is short, fermentation does not proceed, and when the temperature is high and the time is long, the target peptidoglycan derivative may be degraded.

It is preferable that the fermented liquor is filtered with filter paper, diatomaceous earth, 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 100 to 300 g of branched cyclodextrin with respect to 100 g of swallow nests. Ribose and peptide are bound by this branched cyclodextrin.

The suspension with the branched cyclodextrin is preferably stirred.

Protease is added to this suspension. The protease to be added is preferably 0.001 g to 0.3 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. Moreover, stirring is preferably 10 to 30 times per minute. The time is preferably 1 to 6 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.

Furthermore, the obtained reaction product is pulverized by lyophilization and used.

Separating and purifying the desired peptidoglycan 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. In order to increase the purity, it is preferable to repeat the purification step.

For example, the target peptidoglycan derivative can be obtained by separation with a separation carrier or resin and fractionation. As the separation carrier or resin, porous peptidoglycan, silicon oxide compound, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymer and the like whose surface is 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 (manufactured by Mitsubishi Chemical Corporation, HP-20 and HP-21) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH is used as the carrier for the molecular sieve. More preferred is -20 (manufactured by Amersham Pharmacia), silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier.

Of these, Diaion HP-20, 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 HP-20 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 the fraction containing the peptidoglycan derivative and remove the solvent by drying or vacuum drying to obtain the desired peptidoglycan derivative as a powder or a concentrated liquid because the influence of the solvent can be excluded.

In addition, it is preferable to powder this peptidoglycan 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.

Purchased 1kg nest of Indonesian swallows (from swallows) from Nihonbashi Koukiken. This was washed with water and then pulverized by a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain about 1 kg of pulverized product.

In addition, soybeans from Chiba Prefecture were purchased from a soybean factory and pulverized with a pulverizer to obtain approximately 1 kg of pulverized material.

800 g of this swallow's nest suspension and 800 g of pulverized soybean were transferred to a clean stainless steel cylinder and suspended by adding 5 liters of purified water.

This was sterilized by boiling at 95-97 ° C. for 1 hour. These were transferred to a stirring type fermentation tank (FP211) manufactured by Yokogawa Electric Co., Ltd. having a capacity of 100 kg, and 10 liters of sterilized purified water was added.

To this, 10 g of powdered Bacillus natto manufactured by Takahashi Yuzo Laboratory Co., Ltd., which is the natto fungus 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.

The natto fungus solution was added to the agitated fermentation tank and fermented at 40 to 42 ° C. for 48 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.

10 liters of this filtrate was transferred to a clean tank, and 50 g of branched cyclodextrin (Iso Elite) manufactured by Shisui Minato Seiko Co., Ltd. was added thereto and stirred sufficiently.

Further, 5 g of protease M “Amano” SD manufactured by Amano Enzyme was added, and the mixture was heated to 37 ° C. and stirred.

Stirring was carried out for 10 hours at room temperature using a stirrer. This reaction solution was sterilized by boiling for a short time to inactivate 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 Corporation) to obtain 25 g of the intended powder. This was designated as Sample 1.

20 g of the obtained powder of specimen 1 was suspended in 100 mL of purified water and subjected to 500 g of Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) swollen with 7% ethanol. After washing with 700 mL of 7% ethanol, it was further washed with 20% ethanol.

To this, 500 mL of 60% ethanol was added to fractionate the desired peptidoglycan derivative. The obtained fraction was dried with a vacuum dryer. This purification operation was performed 4 times to obtain 10 g of powder as a final purified product. This powder was designated as Sample 2.

Below, the test method regarding the structural analysis of a peptidoglycan 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 in the deuterated dimethyl sulfoxide with the nuclear magnetic resonance apparatus (NMR, the product made from Bruker). As a result of structural analysis, a derivative in which a phenyl group, ribose, cysteine and arginine were bound was detected from Sample 2 and Sample 1.

As for the bond, phenyl group, cysteine and arginine were one molecule each, and ribose was two molecules. Cysteine and arginine were L-type.

As a result of the 600 MHz H-NHR analysis, 0.92, 1.73, 1.84, 2.76, 3.14, 3.23, 3.35, 3.36, 3.43, 3.44, 3 .49, 3.65, 3.67, 3.83, 3.88, 3.94, 4.82, 4.92, 5.03, 5.09, 7.11 and 7.33 ppm. It was.

Furthermore, as a result of C-NMR analysis, 24.2, 25.1, 25.4, 45.7, 50.2, 62.8, 63.1, 68.2, 72.0, 72.1, 75 .6, 75.7, 78.7, 78.8, 80.3, 81.6, 81.9, 95.7, 103.2, 118.9, 132.1, 132.5, 160.7 , 173.1 and 173.8 ppm.

The chart of the analysis result of C-NMR is shown below. (The horizontal axis represents ppm, and the vertical axis represents peak intensity.)

The above analytical values were identical to the peaks of the peptidoglycan derivative synthesized organically, and were identified as the desired peptidoglycan derivative. This derivative contained in Specimen 2 was 99.1%, ie, a purity of 99.1%, while Specimen 1 had a purity of 80.3%.

In addition, when 0.1 g of the obtained derivative powder was dissolved in 10 mL of purified water, generation of hydrogen gas was observed. As a result of quantification by gas chromatography (manufactured by Shimadzu Corporation), a hydrogen gas concentration of 1.6 ppm was exhibited.

The confirmation test of anti-inflammatory action using human-derived leukocytes is described below.
(Test Example 2)

Heparinized blood collected from 5 healthy humans (2 males and 3 females, age 25-61 years) was aseptically collected under the informed concept under a doctor. White blood cells were collected from the collected blood by centrifugation using Ficoll. The leukocytes were suspended in RPMI-1640 medium (manufactured by Wako Pure Chemical Industries, Ltd.) and cultured in a carbon dioxide incubator (MG-70C, manufactured by Taitec) at 37 ° C. and 5% carbon dioxide. The cells were seeded on a 96-well microplate (manufactured by Falcon), and a solution of LPS (lipopolysaccharide, manufactured by Wako Pure Chemical Industries) was added thereto. This final concentration was 0.1 mg / mL.

Specimen 2 as the test substance and loxofen sodium of LSAIDs (Loxonin, Daiichi Sankyo) as the test substance are both suspended in physiological saline and diluted to a final concentration of 0.1 mg / mL. Added. Note that physiological saline was used as a solvent control. This was cultured at 37 ° C. for 3 days, and the number of viable cells was counted under a microscope. Furthermore, the amount of TNF alpha (Funakoshi, IDELISA ELISA kit) and the amount of prostaglandin E2 collected by collecting the supernatant were colorimetrically determined by ELISA (Cosmo Bio Inc.).

As a result, as a result of obtaining the ratio by adding the sample 2 with the number of cells of the solvent control as 100%, it increased to 133% by adding the sample 2. On the other hand, with loxofen sodium, it was 111%, and specimen 2 was superior in protecting leukocytes.

The amount of TNF alpha was found to be 26% with the addition of Specimen 2 as a result of obtaining the ratio by the addition of Specimen 2 with the solvent control as 100%. On the other hand, with loxofen sodium, it was 44%, and specimen 2 was more excellent in suppressing TNF alpha.

The amount of prostaglandin E2 was reduced to 23% by the addition of specimen 2 as a result of obtaining the ratio by the addition of specimen 2 with the solvent control as 100%. On the other hand, with loxofen sodium, it was 40%, and the specimen 2 was more excellent in suppressing the amount of prostaglandin E2. Further, this derivative was considered to be highly safe from the result that it did not damage human-derived leukocytes.

The confirmation test for keratin production using human-derived skin cells is described below.
(Test Example 3)

Human skin-derived first representative skin cultured cells purchased from Cosmo Bio Inc. were used. The cells were suspended in a dedicated culture solution and pre-cultured to grow the cells. The cells were cultured in a carbon dioxide incubator at 37 ° C. under 5% carbon dioxide gas. Thereafter, the cells in the growth phase were detached with a trypsin-containing medium and used for the experiment. Viable cell numbers were counted under a microscope by trypan blue dye exclusion. The number of cells was adjusted to 1000 per mL, seeded in 5 mL culture dishes, and further cultured at 37 ° C. under 5% carbon dioxide gas. This was irradiated with ultraviolet rays by an ultraviolet irradiation device (Loctite, output 88 MH) to damage the cells. Irradiation was carried out for 1 hour with the petri dish lid removed.

Skin cells were damaged by this ultraviolet irradiation, and recovery from this damage was tested. In addition, this method is a method implemented for evaluation of the test substance with respect to skin.

Specimen 2 as a test substance and human EGF (manufactured by Funakoshi) as a test substance were both suspended in physiological saline, diluted and added to a final concentration of 0.1 mg / mL. Note that physiological saline was used as a solvent control. This was cultured at 37 ° C. for 3 days, and the number of viable cells was counted under a microscope. Further, the cells were dispersed in purified water, and a cell dispersion was obtained using an ultrasonic crusher. The amount of keratin contained in the cell dispersion was quantified by ELISA (Cosmo Bio Inc.).

As a result, the ratio by adding the sample 2 was determined with the number of cells of the solvent control as 100%. On the other hand, EGF is 177%. Specimen 2 was superior in skin cell proliferation action. The amount of keratin was increased to 381% by adding Sample 2 with the solvent control value being 100%. On the other hand, EGF was 240%, and Sample 2 was superior in keratin production. The hydrogen gas concentration in the solvent was 1.6 ppm. In addition, this derivative was considered to be highly safe from the results that the number of cells was recovered without causing damage to human-derived skin cells.

The peptidoglycan derivative obtained in the present invention exhibits an anti-inflammatory action and has few side effects. Therefore, the peptidoglycan derivative is used for treatment and prevention and can improve the QOL of the people.

The peptidoglycan derivative obtained in the present invention is also used as a cosmetic for skin improvement and contributes to the development of the cosmetic industry.

Claims (1)

A peptidoglycan derivative exhibiting an anti-inflammatory action represented by the following formula (1).