JP6010073B2 - Resveratrol derivative producing hydrogen gas and exhibiting keratin production action and method for producing the same - Google Patents

Description

The present invention relates to a resveratrol derivative that produces hydrogen gas and exhibits a keratin producing action and a method for producing the same.

Keratin is an important protein that constitutes cells. In the skin, it works to increase the elasticity of the skin, and the hair is given elasticity.

Keratin constitutes a stratum corneum barrier layer in the stratum corneum of the skin and protects the skin from bacterial infection. It is a functional protein that functions to control inflammation. However, the keratin content decreases with age, which causes a decrease in skin elasticity and wrinkles.

Although there is a diene compound used in human epidermal cell repair as an invention for producing and maintaining keratin, and pharmaceutical and cosmetic compositions containing it, it is a chemical substance and has not yet been identified as a specific substance. Use is limited (see, for example, Patent Document 1).

Further, as an invention relating to keratin production, for example, there is a hypoglycemic and / or anti-obesity composition containing an acacia bark-derived product (see, for example, Patent Document 2).

However, its application is limited.

Japanese Patent Application No. 2011-535127 Japanese Patent Application No. 2012-5292

The keratin production action by the existing substances is mild, and there is a problem that the industrial use is limited, and the chemically synthesized substance has a problem in safety and the use is limited.

Therefore, a natural product that exhibits an excellent keratin production effect 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 resveratrol derivative that produces hydrogen gas represented by the following formula (1) and has a keratin producing action.

In order to achieve the above object, the invention described in claim 2 relates to a method for producing a resveratrol derivative that produces hydrogen gas represented by the formula (1) described in claim 1 and has a keratin-producing action. It is.

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

The resveratrol derivative according to claim 1 produces hydrogen gas and is excellent in keratin production action.

According to the manufacturing method of Claim 2, a resveratrol derivative can be manufactured efficiently.

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

The resveratrol derivative that produces hydrogen gas and exhibits a keratin producing action has a structure represented by the following formula (1).

As shown in the above formula (1), one molecule of thymine is bonded to two molecules of resveratrol.

Resveratrol is a derivative of cinnamic acid among organic acids, and 3,4,5-trihydroxy-trans-stilbene or trans-1,2- (3,4,5-trihydroxydiphenyl) ethylene and Called.

Resveratrol is contained in plants such as camellia, cinnamon bark, blueberries and acai. Of these, acai is abundant. In particular, it is preferable because the acai fermented extract obtained by fermenting acai fruit together with rice bran with benichow fungus is abundant.

In this resveratrol derivative, two molecules of resveratrol are bound. That is, the hydroxyl group at the 5-position is bonded to the hydroxyl group at the 3-position of another molecule.

In addition, one molecule of thymine is bound to resveratrol. In this bond, the phenolic hydroxyl group of resveratrol is bonded to the nitrogen molecule of thymine through oxygen.

That is, this resveratrol derivative is an organic compound containing a nucleobase. All of these substances are naturally derived and their safety has been confirmed.

In addition, when a person comes into contact with or drinks an excessive amount of this resveratrol derivative, it is decomposed by enzymes such as esterase in the body and decomposed into resveratrol and thymine, which is highly safe. .

This resveratrol derivative is preferable because it is easily decomposed by microorganisms in the soil, has no burden on the environment, and does not accumulate.

This resveratrol derivative passes through the cell membrane, and further passes through the nuclear membrane and acts on the gene, so that the action is preferable because it is direct and efficient. This resveratrol derivative activates and amplifies the gene of keratinase, so that keratinase is induced and keratin production increases.

In addition, this resveratrol derivative is preferable because it exhibits excellent reducing power and generates hydrogen gas. That is, when this resveratrol derivative is dissolved in water, it generates hydrogen gas, maintains the reducing power, and protects the cells from oxidation. Moreover, it is preferable to exhibit an action of removing active oxygen.

This resveratrol derivative works on skin cells and suppresses the degradation of keratin, which is preferable because keratin is maintained. Moreover, it is preferable that the generated hydrogen gas maintain keratin.

Furthermore, this resveratrol derivative activates the proliferation of skin epidermal cells. The proliferation of skin cells by this resveratrol derivative is due to a stimulating action at the gene level.

This resveratrol derivative is preferred because it activates skin cell turnover and leads to proliferation of skin cells. At this time, the generated hydrogen gas is preferable because it protects cells.

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

Used as pharmaceuticals, parenteral preparations such as injections, oral preparations and coatings, and quasi-drugs used in tablets, capsules, drinks, soaps, coatings, gels, toothpastes, etc. Is done.

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 health food and beauty food for the purpose of keratin production and beauty. Moreover, as a health functional food, it is preferable to use it for a nutrition functional food or a food for specified health.

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 maintaining keratin production and skin health.

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 and the like.

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

The resulting cosmetic is used for keratin production to improve wrinkles, protect against tarmi, and form a skin barrier layer for atopic dermatitis.

Next, a method for producing a resveratrol derivative represented by the formula (1), which produces hydrogen gas comprising a step of fermenting acai fruit and rice brance with Benikouji fungus manufactured by Kurisu Honpo and exhibiting a keratin producing action, will be described. .

The resveratrol derivative mentioned here is a derivative represented by the above formula (1) from two resveratrol molecules and one thymine molecule. This resveratrol derivative is highly safe because it is decomposed and excreted by enzymes in vivo.

This resveratrol derivative resveratrol is preferable because it exists in nature, has abundant food experience, and is recognized as safe.

This derivative acts directly on skin cell genes and exerts keratin production.

This production method comprises a step of fermenting with acai fruit, rice bran powder and Benikouji fungus made by Kurisu Honpo.

The raw materials are acai fruit, rice bran powder and Benikouji fungus from Kurisu Honpo.

Acai is the scientific name Europe Oleracea and the Japanese name is Wakaba cabbage palm. It is a palm family plant native to Brazil. Its fruits are similar to blueberries and are used as food.

Acai fruits contain polyphenols and resveratrol, which is preferable as a raw material for this derivative.

Acai fruit may come from any country such as Brazil or other South American or American. In addition, those produced with low pesticides or reduced pesticides are preferred.

Acai fruits are preferably dried and pulverized, and autoclaving before fermentation is preferable because fermentation can be performed smoothly.

A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

The rice bran powder used as a raw material can be used from rice in any production area such as Japanese, Chinese, American, and Russian, but is preferably made in Japan with reliable traceability and a clear producer.

Of these, rice cultivated organically or without agrochemicals is more preferred because it does not contain harmful agrochemicals or metals.

In use, KOMENUKA is a free-form mill, super-free mill, sample mill, goblin, super-clean mill, micros, vacuum dryer from Toyo Riko 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.

Furthermore, it is preferable that the acai fruit and rice bran be sterilized after being pulverized by an autoclave or the like because propagation of germs can be prevented.

This is fermented by Benikouji fungi from Kurisu Honpo. The resveratrol derivative is separated by the fermentation by Beniculobacterium, and absorption is also promoted.

The Benikouji fungus made by Benito Honpo to be used is a filamentous fungus of the scientific name Monasuc purpureus and has long been used in fermented foods such as red sake and tofu in Japan, China and Taiwan. In addition, bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia in China and Taiwan are used. Benikouji fungus made by Kurisu Honpo has excellent fermentation efficiency.

As for each addition amount regarding the said fermentation, 0.0001-0.009 weight is preferable with respect to 1 weight of said mixtures. It is preferable to pre-cultivate Benikouji fungus made by Kurisu Honpo before fermentation because it shortens the initial time of fermentation and shortens the fermentation time.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

Moreover, this fermentation is heated at 39-45 degreeC, and fermentation is performed for 20 days from 1 day.

Resveratrol derivatives are formed by this fermentation process.

Moreover, since the product is easy to isolate | separate, it is preferable to ultrasonically treat the obtained fermented material. Moreover, it is preferable to concentrate by freeze drying or the like because the following steps can be performed in a short time.

Separating and purifying the desired resveratrol derivative from the reduction reaction product is preferable because it can reduce intake 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 intended resveratrol derivative is obtained by separating and separating with a separating 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 is preferably 1 to 35 times the weight of the extract from the viewpoint of separation efficiency, and more preferably 4 to 25 times the amount. 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 the fraction containing the resveratrol derivative and remove the solvent by drying or vacuum drying to obtain the desired resveratrol derivative as a powder or a concentrated liquid because the influence of the solvent can be excluded.

Moreover, it is preferable to carry out the final extraction with fats and oils used for edible oils and cosmetics because the obtained resveratrol derivative is stably maintained. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

In addition, it is preferable to powder this resveratrol 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.

Acai fruits cultivated with reduced pesticides in Brazil were used. After collecting the fruit, it is washed with tap water, dried in the sun, and pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.1 kg of dry powder pulverized product of Acai fruit It was.

Rice bran obtained from Niigata rice was used in a mixer (Cuisinart) to obtain 1 kg of rice bran pulverized material. The acai fruit and rice bran powder were subjected to autoclaving and sterilized at 121 ° C. for 20 minutes.

These were put into a clean fermentation tank (sterilized round 40-liter tank for fermentation), and 11 kg of sterilized tap water was added and stirred.

12 g of Benikouji fungus made by Kurisu Honpo was added and fermented at 39 ° C. for 7 days.

This fermented product was warmed and ethanol was added to obtain 402 g of a target resveratrol derivative-containing extract.

1 L of 5% ethanol-containing purified water was added to 300 g of the aforementioned resveratrol derivative-containing extract, and this was applied to a column packed with 500 g of Diaion (manufactured by Mitsubishi Chemical) suspended in 5% ethanol solution.

2 L of 5% ethanol solution was added thereto for cleaning, and 1 L of 60% ethanol solution was added to elute the desired resveratrol derivative for purification. The purified resveratrol derivative was distilled under reduced pressure to remove the ethanol portion to obtain an aqueous solution. This was designated as Resveratrol derivative specimen 1.

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

The specimen 1 obtained as described above was dissolved in ethanol 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 Bruker make, AC-250). As a result of structural analysis, resveratrol and base thymine were detected from specimen 1. In addition, binding of resveratrol 2 molecules and binding to thymine were confirmed. Further structural analysis identified the structure of this resveratrol derivative.

That is, two molecules of resveratrol were bound. The hydroxyl group at the 5-position and the hydroxyl group at the 3-position of another molecule were bonded. In addition, one molecule of thymine was bound to resveratrol. This bond was a bond to the 9th-position nitrogen molecule of thymine with a phenolic hydroxyl group at the 4-position of resveratrol, and was bonded via oxygen.

The confirmation test using human skin epidermal cells is described below.
(Test Example 2)

Human skin epidermis cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, culturing was performed using a 5% fetal calf serum-containing MEM medium (manufactured by Sigma). 1000 cells were seeded in a 35 mm culture dish and cultured at 37 ° C. in 5% carbon dioxide gas. To this, specimen 1 obtained in Example 1 above and EGF (epidermal growth factor) as a control at a final concentration of 0.1 mg / ml were added. This was cultured for 48 hours.

After detaching the cells, the number of cells was counted, a cell suspension was prepared, and the amount of keratin in the cells was measured by ELISA (Wako Pure Chemical Industries). In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Specimen 1 increased the number of epidermal cells to 542% as an average value compared to the control group. EGF increased by 180%, and specimen 1 was superior.

The amount of keratin was increased to 423% by Specimen 1 compared to the control group. EGF was 177%, and the increase due to specimen 1 was remarkable. The cells were not damaged and the safety was confirmed.

In the following, the confirmation test of hydrogen gas generation is described.
(Test Example 3)
Hydrogen gas production was tested by gas chromatography. That is, 100 mg of Sample 1 was placed in a sealed container together with 10 mL of purified water, and the concentration of hydrogen gas produced in the air layer was measured 1 hour after dissolution.

Gas chromatography SGHA-C1 equipped with a semiconductor gas sensor manufactured by Taiyo Nissan was used. The detection limit of hydrogen gas was 0.05 ppm.

As a result, 22 ppm of hydrogen gas was detected from Sample 1 after 1 hour. This value exceeded the atmospheric concentration, and hydrogen gas generation was confirmed.

The binding confirmation test using DNA derived from human skin epidermis cells is described below.
(Test Example 4)
Human skin epidermis cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using a 5% fetal calf serum-containing MEM medium (manufactured by Sigma) were obtained.

DNA was collected from the cells using a DNA extraction kit (Wako Pure Chemical Industries, Ltd.). The obtained DNA was applied to a sensor of a Biacore system (manufactured by GE Healthcare, type 4000).

Similarly, the interaction between DNA and DNA was measured by the surface plasmon resonance method using the obtained DNA as a ligand.

The sample 1 solution was diluted with physiological saline. Saline was used as a solvent control.

As a result, the binding property of Sample 1 was 255% of the solvent control, and an increase in DNA binding property was observed.

From this result, it was considered that Specimen 1 caused activation of DNA reaction in human skin epidermis cells.

The resveratrol derivative obtained in the present invention protects cellular tissues by increasing keratin, and contributes to maintaining the health of the nation by protecting the tissues.

The resveratrol derivative obtained in the present invention has an action to improve skin epidermal cells and keratin, and therefore contributes to improvement of those suffering from inflammation, atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetic industry. .

Since the resveratrol derivative obtained by the present invention can be used as food, it contributes to the development of the food industry.

Claims (2)

A resveratrol derivative that produces hydrogen gas represented by the following formula (1) and exhibits a keratin production action.

The method for producing a resveratrol derivative represented by the formula (1) according to claim 1, which produces hydrogen gas comprising a step of fermenting acai fruit and rice bran with Benikouji fungus manufactured by Kurisu Honpo, and exhibits a keratin producing action. .