JP6048905B2 - Uronic acid derivatives exhibiting an action to increase the expression level of longevity gene Sirt1 applying curing culture - Google Patents

Description

The present invention relates to a uronic acid derivative exhibiting an action of increasing the expression level of the longevity gene Sirt1 to which a curing culture is applied.

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 target was to increase the expression level of Sirt1 , which is one of the longevity genes.

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 is highly safe, but has a disadvantage that the action of increasing the expression level of the longevity gene Sirt1 is mild and has a weak effect, and its industrial use is limited.

Japanese Patent Application No. 2013-518563

As described above, the effect of increasing the expression level 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. The use is limited.

Therefore, a natural product that exhibits an effect of increasing the expression level of the longevity gene Sirt1 , which has excellent side effects and is weak, is desired.

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

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 expression level increasing effect of the longevity gene Sirt1 is exhibited.

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

The uronic acid derivative exhibiting the effect of increasing the expression level of the longevity gene 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 increasing the expression level 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.

These two actions work synergistically to increase and maintain the expression of the longevity gene Sirt1, and increase 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 aims to increase the expression level of the longevity gene Sirt1 , and is used for health foods and foods for the purpose of anti-aging. 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.

It can be used together with an agent for increasing the expression level of an interface, a solvent, a thickener, an excipient and the like according to a conventional method as a cosmetic. 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.

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 increase in the expression level of the longevity gene Sirt1 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.

The confirmation test of the expression level increasing action of the longevity gene Sirt1 using human neurons will be described below.
(Test Example 4 )

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 an action of increasing the expression level of the longevity gene Sirt1 and has few side effects. Therefore, it can improve national QOL as an anti-inflammatory agent and reduce medical costs.

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 (1)

A uronic acid derivative exhibiting an action of increasing the expression level of the longevity gene Sirt1 represented by the following formula (1).