JP2015117212A - Uronic acid derivative which shows longevity gene sirt1 activating action adapting health care culture, and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an uronic acid derivative which shows longevity gene Sirt1 activating action adapting health care culture, and to provide a production method thereof.SOLUTION: The target uronic acid derivative comprises a tripeptide consisting of arginine, threonine, and asparagine, and uronic acid. The uronic acid is bound to the arginine of the tripeptide and shows longevity gene Sirt1 activating action. This production method comprises adding branched cyclodextrin into the fermented solution obtained by fermenting unpolished rice, black rice, red-kerneled rice, barley, rice cake wheat, pigeon wheat, foxtail millet, Japanese millet, millet, Sorghum bicolor, soybean, black soybean, adzuki bean, and corn with Bacillus natto, performing protease treatment, subsequently performing alkali reduction of the filtered filtrate.

Description

The present invention relates to a uronic acid derivative exhibiting a longevity gene Sirt1 activating action applying a curing culture and a method for producing the same.

Curative culture is an oriental medical and scientific idea, a multiple theory related to the foundation of life, has a long history, and has extensive experience in use.

A medicinal candy is a food-related technique that uses a culture of healing. On the other hand, there is a traditional beauty method using a curing culture, which is used for plant extracts and fermentation raw materials.

However, it is difficult to medically examine the curing culture and apply it to industry. The reason is that specific components cannot be identified and their functions are diverse.

Therefore, the traditional fermentation method was used in terms of applying curing culture. Furthermore, we decided to analyze the curing culture at the genetic level. Since the longevity gene is involved in the basis of life activity, the activation of Sirt1, which is one of the longevity genes, was targeted.

For example, as an invention using a longevity gene, there is an invention of a resveratrol-containing composition and a method of use, but the range of use is limited (see, for example, Patent Document 1).

In addition, the chemically synthesized components have a problem that side effects are observed.

On the other hand, a substance derived from a natural product has high safety, but has a disadvantage that the longevity gene Sirt1 activation is mild and its effect is weak, and its industrial use is limited.

Japanese Patent Application No. 2013-518563

As described above, the activation of the longevity gene Sirt1 by existing natural products is mild, and there is a problem that its use in industry is limited, and there is a problem in safety with chemically synthesized substances, Use is limited.

Therefore, a natural product exhibiting an excellent longevity gene Sirt1 activation action with weak side effects is desired. Furthermore, a production method for efficiently producing a substance exhibiting the longevity gene Sirt1 activation action is desired.

In order to achieve the above object, the invention described in claim 1 relates to a uronic acid derivative exhibiting a longevity gene Sirt1 activating action to which a curing culture represented by the following formula (1) is applied.

In order to achieve the above object, the invention described in claim 2 relates to a method for producing a uronic acid derivative exhibiting a longevity gene Sirt1 activating action by applying a curing culture.

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

According to the uronic acid derivative according to claim 1, an excellent longevity gene Sirt1 activation action is exhibited.

According to the manufacturing method of Claim 2, the uronic acid derivative which exhibits a longevity gene Sirt1 activation effect | action efficiently can be manufactured.

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

The uronic acid derivative exhibiting the longevity gene activation action has a structure represented by the following formula (1).

The uronic acid derivative here is a conjugate of uronic acid in which a peptide in which two molecules of uronic acid are bound to one tripeptide molecule composed of uronic acid and arginine, threonine, and asparagine is bound.

Arginine, threonine, asparagine and uronic acid are all contained in natural plants, and their safety has been confirmed.

The tripeptide is composed of arginine, threonine, and asparagine, the N-terminal side is arginine, the center is threonine, and the C-terminal side is asparagine, and they are bound by peptide bonds.

All of these amino acids are in L form. All of these amino acids are components present in the body, and their safety has been confirmed.

The carboxyl group of uronic acid is peptide-bonded to the amino group of the guanidino group of arginine.

This uronic acid derivative is highly water-soluble and, on the other hand, also has an affinity for alcohol, it exhibits solubility in ethanol, glycols, glycerins, etc. and is industrially easy to use.

This uronic acid derivative has two action mechanisms for activation of the longevity gene Sirt1.

One is a case where it acts directly on the promoter site of the longevity gene Sirt1 to act as a promoter and promotes the expression of Sirt1. Sirt1 forms a sirtuin family, and abundant promoters exist in the vicinity of the gene.

This uronic acid derivative reacts with the promoter at the peptide site and maintains its expression by uronic acid. However, its function is transient and not a strong bond such as a covalent bond.

The other is suppression of degradation of the longevity gene Sirt1. Its function is by stabilizing the chromatin moiety.

The synergistic action of these two actions activates and maintains the longevity gene Sirt1, and increases the longevity gene Sirt1.

In addition, this uronic acid derivative is decomposed by peptidases and esterases localized in the cells to be decomposed into peptides and uronic acids, so there is no persistence and safety is high.

When the obtained uronic acid derivative is used as a pharmaceutical material, it is preferable to separate and purify the target uronic acid derivative because the purity of the target uronic acid derivative is increased and impurities can be removed.

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.

As a food preparation, it is intended to activate the longevity gene Sirt1, and is used for health foods and foods for anti-aging purposes. 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 feed or supplement for the purpose of anti-aging.

As a cosmetic, it can be used together with surfactants, solvents, thickeners, excipients 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 and the like.

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

The obtained cosmetic increases the longevity gene Sirt1, exhibits skin function by anti-aging, and is used for preventing wrinkles and improving tarmi.

Next, branched cyclodextrin is added to fermentation broth fermented brown rice, black rice, red rice, barley, glutinous wheat, wheat, potatoes, rice cakes, rice cakes, oysters, soybeans, black beans, red beans and corn with natto bacteria A method for producing a uronic acid derivative exhibiting the longevity gene Sirt1 activation action comprising the step of subjecting the filtrate to alkali reduction after the protease treatment will be described.

The uronic acid derivative here is a peptide uronic acid conjugate in which two molecules of uronic acid are bound to one tripeptide molecule composed of uronic acid and arginine, threonine, and asparagine.

Arginine, threonine, asparagine and uronic acid are all contained in natural plants, and their safety has been confirmed.

The tripeptide is composed of arginine, threonine, and asparagine, the N-terminal side is arginine, the center is threonine, and the C-terminal side is asparagine, and they are bound by peptide bonds.

All of these amino acids are in L form. All of these amino acids are components present in the body, and their safety has been confirmed.

The carboxyl group of uronic acid is peptide-bonded to the amino group of the guanidino group of arginine.

This uronic acid derivative is highly water-soluble and, on the other hand, also has an affinity for alcohol, it exhibits solubility in ethanol, glycols, glycerins, etc. and is industrially easy to use.

This uronic acid derivative exhibits the longevity gene Sirt1 activation action.

The raw materials are brown rice, black rice, red rice, barley, glutinous wheat, wheat wheat, rice bran, rice bran, rice bran, oysters, soybeans, black beans, red beans and corn. These grains are preferably organically grown. That is, fermentation is efficient, and the desired uronic acid derivative can be obtained in high yield.

Moreover, although any country may be sufficient as a producing country, the thing from Japan is preferable from the point of local production for local consumption.

These cereals are washed with water and then chopped by a cutter or a pulverizer to obtain a pulverized product. The pulverized product is dried to obtain a cereal powder.

These cereal powders contain uronic acids such as uronic acid and cinnamic acid in addition to carbohydrates, lipids, proteins and peptide components.

These cereal powders are suspended with the addition of clean water. 10 to 20 liters of water is added to 100 g of cereal powder and stirred.

These grains are sterilized by boiling and added to the fermentation tank.

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 49 ° C. for 12 to 36 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 uronic 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 0.5 to 5 g per 10 g of cereal. This branched cyclodextrin encapsulates the peptide-uronic acid conjugate in the grain.

The suspension with the branched cyclodextrin is preferably stirred.

Protease is added to the suspension of cereal and branched cyclodextrin. 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.

The protease to be added is preferably 0.02 to 0.1 g per gram of cereal. 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, 30-45 degreeC is preferable. Moreover, stirring is preferably 10 to 30 times per minute.

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 subjected to alkali reduction treatment. The alkali reduction treatment is preferably performed by an alkali reduction device or an alkali reduction water conditioner.

For example, a continuous generator of alkaline reduced water / strongly oxidized water “Protech ATX-501” manufactured by Zemaitis, an alkali reduced water production device “Techno Super 502” manufactured by NCI, “Mineria CE-212” manufactured by Maltaca, manufactured by Crescent It is more preferable to use devices such as “Acura Blue” and “Mineral Reduction Water Conditioner” manufactured by Nippon Kosen Co., Ltd.

By electrolysis, a conjugate of the desired uronic acid derivative and peptide is obtained as a solution from the cathode side. This alkali reduction results in binding of uronic acid and peptide.

It is preferable because the reaction is enhanced by repeating the alkali reduction treatment 2 to 10 times. The obtained conjugate is powdered by lyophilization and used.

Separating and purifying the target uronic acid derivative from the reduction reaction product is preferable because 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 target uronic acid derivative can be 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 1 to 30 times, more preferably 5 to 20 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 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.

It is preferable to collect a fraction containing a uronic acid derivative and remove the solvent by drying or vacuum drying to obtain the target uronic 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 uronic 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.

Organically grown or reduced pesticide-grown brown rice, black rice and red rice from Niigata Prefecture, barley and glutinous wheat from Yamagata Prefecture, wheat and wheat from Tottori Prefecture, straw, rice cake and straw from Shizuoka Prefecture, Taka from Miyazaki Prefecture Millet, soybeans from Ibaraki Prefecture, black beans from Kyoto Prefecture, red beans from Mie Prefecture, and corn from Hokkaido were purchased and used.

These were washed with water and then pulverized together with purified water in a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 9 kg of pulverized product.

The pulverized product was dried with a drier to obtain a cereal powder. 8.5 kg of this cereal powder was transferred to a clean stainless steel cylinder, and 15 L of purified water was added and suspended.

These were sterilized by boiling at 95 ° C. for 1 hour. These were transferred to an agitation type fermentation tank (FP211) manufactured by Yokogawa Electric Co., Ltd. with a capacity of 80 kg and fermented at 41 ° C. for 24 hours.

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

To this filtrate, 240 g of branched cyclodextrin manufactured by Shimizu Minato Sugar Co., Ltd. was added and stirred.

Furthermore, 20 g of protease M “Amano” SD manufactured by Amano Enzyme was added, and the mixture was heated to 38 ° C. and stirred.

Stirring was carried out for 4 hours at room temperature using a stirrer. The obtained reaction solution was subjected to suction filtration with a filter paper of Toyo filter paper to obtain a filtrate.

The obtained reaction solution was subjected to Pearl Water DX-7000 and electrolyzed to obtain an alkali-reduced solution from the cathode side.

This solution was lyophilized to obtain 230 g of the desired powder. This was designated as Sample 1. This specimen 1 was light yellow.

2 L of 10% ethanol-containing purified water was added to 100 g of the aforementioned sample 1 powder, and 500 g of Diaion (manufactured by Mitsubishi Chemical) was suspended in a 5% ethanol solution and packed in a column.

4 L of 5% ethanol solution was added thereto for cleaning, and then 1 L of 80% ethanol solution was added to elute the desired uronic acid derivative. The purified uronic acid derivative was subjected to distillation under reduced pressure to remove the ethanol portion, and this was used as Sample 2. Sample 2 was tasteless and odorless and transparent and water-soluble.

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

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

Furthermore, it analyzed with the nuclear magnetic resonance apparatus (NMR, the Bruker make, AC-250). As a result of structural analysis, a conjugate in which uronic acid, arginine, threonine, and asparagine were bound was detected from specimen 2.

Arginine, threonine, and asparagine were identified by an amino acid analyzer (manufactured by Shimadzu Corporation).

The confirmation test for the longevity gene Sirt1 activation using human skin cells is described below.
(Test Example 2)

This test is a cytomolecular method in which a specimen is added to human-derived skin cells and cultured, and the RNA amount of the longevity gene Sirt1 is analyzed by the RT-PCR method. These methods are established as general analytical methods.

That is, normal human-derived skin cells were cultured in a dedicated culture solution. To this, 0.1 mg of Specimen 2, uronic acid, and arginine obtained in Example 1 was added in a 5% ethanol-containing PBS solution, and cultured at 37 ° C. for 24 hours. A solvent control was set as a control group.

After counting the number of cells, the cell suspension was sonicated to prepare a cell suspension. An RNA fraction was collected from this cell solution using an RNA extraction kit (Funakoshi).

This RNA fraction was analyzed by the electromigration method using the longevity gene Sirt1 as a probe by the RT-PCR method, and the longevity gene Sirt1 content was quantified.

As a result, the longevity gene Sirt1 was 452% as compared with the solvent control, and a clear increase was observed by the treatment of specimen 2. On the other hand, it was 120% when uronic acid was added, and 103% when arginine was added, which was similar to the solvent control.

The elastin degradation test using human skin cells is described below.
(Test Example 3)

Purified elastin was purchased from Sigma. Elastin was dissolved in Tris buffer (pH 7.4). This was treated with elastase to decompose elastin, and the degradation rate of elastin was counted using the change in absorbance at 280 nm as an index.

Under this condition, a 0.1 mg / mL solution of Specimen 2 was added and the degradation rate of elastin was measured.

As a result, when specimen 2 was added as compared with the solvent control, the degradation rate of elastin decreased to 55%. Specimen 2 showed an elastin degradation inhibitory effect.

Below, the confirmation test of longevity gene Sirt1 activation using a human nerve cell is described.
(Test Example 2)

In this test, a sample was added to a human-derived nerve cell (manufactured by Kurabo Industries) and cultured, and the RNA amount of the longevity gene Sirt1 was analyzed.

That is, normal human-derived nerve cells were cultured in a dedicated culture solution. To this, 0.1 mg of the specimen 2 obtained in Example 1 was added in a PBS solution containing 5% ethanol, and cultured at 37 ° C. for 40 hours. A solvent control was set as a control group.

After counting the number of cells, the cell suspension was sonicated to prepare a cell suspension. An RNA fraction was collected from this cell solution using an RNA extraction kit (Funakoshi).

This RNA fraction was analyzed by the electromigration method using the longevity gene Sirt1 as a probe by the RT-PCR method, and the longevity gene Sirt1 content was quantified.

As a result, the longevity gene Sirt1 was 503% compared to the solvent control by the treatment of the specimen 2, and an obvious increase was observed.

The uronic acid derivative obtained by the present invention exhibits the longevity gene Sirt1 activating action and has few side effects. Therefore, it can improve national QOL as an anti-inflammatory agent and reduce medical costs.

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

The uronic acid derivative obtained in the present invention is used as a cosmetic for improving wrinkles and spots and contributes to the development of the cosmetic industry.

Claims (2)

The uronic acid derivative which exhibits the longevity gene Sirt1 activation action shown by following formula (1).

Brown rice, black rice, red rice, barley, glutinous wheat, hato barley, rice cake, rice cake, rice bran, oysters, soybeans, black beans, red beans and corn fermented with natto bacteria to add a branched cyclodextrin to protease The manufacturing method of the uronic acid derivative which exhibits the longevity gene Sirt1 activation action which consists of the process of carrying out the alkali reduction of the filtrate which processed and filtered.