JP2016088858A - Resveratrol derivative that generates hydrogen and exhibits keratin producing action and method for producing the derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a resveratrol derivative that generates hydrogen and exhibits keratin producing action, which is useful as a cosmetic, a food preparation, and a medicine, and which is, especially when used in cosmetic, expected to improve degradation of sebum, moisture holding power of skin, and resilience of skin, and to provide a method for producing the derivative.SOLUTION: Provided is a resveratrol derivative having a structure represented by the following formula (1) where four molecules of resveratrol are bonded, the resveratrol being obtained by subjecting a fermentation liquid to a protease treatment, followed by separation and purification by an ion exchange resin and the like, the fermentation liquid being obtained by adding Bacillus natto and Monascus purpureus to sprout-containing grape seeds and soybean powder and fermenting the mixture.EFFECT: The resveratrol derivative shows an amphiphilic property including hydrophobicity inside the molecule and hydrophilicity outside the molecule. The hydroxy group at 4-position of each resveratrol is free, and these hydroxy groups generate hydrogen, reduces cells, passes through cell membranes, activates genes of a keratin generating enzyme to increase production of keratin, suppresses decomposition of keratin, and activates proliferation of epithelial cells.SELECTED DRAWING: None

Description

The present invention relates to a resveratrol derivative that generates hydrogen 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 keratin barrier 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 are diene compounds used in human epithelial cell repair, and pharmaceutical and cosmetic compositions containing them as inventions for producing and maintaining keratin, they are chemical substances and have not yet been identified as specific substances. Use is limited (see, for example, Patent Document 2).

Moreover, 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 1), but its application is limited.

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

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 which generates hydrogen 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 which generates hydrogen and has a keratin producing action.

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

The resveratrol derivative according to claim 1 generates hydrogen 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 generates hydrogen and exhibits keratin production has a structure represented by the following formula (1).

As shown in the above formula (1), it has a structure in which four molecules of resveratrol are bonded and has a hydroxyl group to generate hydrogen gas.

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

Resveratrol is contained in plants such as grapes, cinnamon bark, blueberries and acai.

Of these, grape seeds are abundant. Resveratrol has an excellent antioxidant effect and has been confirmed to affect genes.

In this resveratrol derivative, 4 molecules of resveratrol are bonded via the hydroxyl groups at the 3rd and 5th positions, respectively. Due to this bond, the inside of the molecule becomes hydrophobic, while the outside is hydrophilic and thus exhibits amphipathic properties. These hydroxyl groups promote the generation of hydrogen.

The 4-position hydroxyl group of each resveratrol of this resveratrol derivative is free, this hydroxyl group generates hydrogen, and activates the hydroxylation reaction of keratin synthase. Generation of hydrogen is preferable because the cells are stabilized.

This resveratrol derivative can be synthesized organically and used as a standard product. The structure was analyzed by H-NMR, and chemical shift peaks were 1.14, 2.26 to 2.63, 3.9 to 4.3, 5.6 to 6.77, and further 7. Presented at 13 ppm.

That is, this resveratrol derivative is naturally derived and preferable because its safety has been confirmed. Furthermore, when a person comes into contact with or drinks an excessive amount of this resveratrol derivative, it is decomposed by an enzyme such as esterase in the body and is decomposed into resveratrol molecules, 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 is preferable because it reduces the cell by generating hydrogen and passes through the cell membrane, and further passes through the nuclear membrane to act on the gene, so that its action is direct and efficient.

This resveratrol derivative activates and amplifies the gene of keratinase by generating hydrogen, thereby inducing keratinase and increasing keratin production.

This resveratrol derivative works on skin cells and suppresses the degradation of keratin, which is preferable because keratin is maintained.

Furthermore, this resveratrol derivative activates the proliferation of skin epithelial 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 stabilizes cells by generating hydrogen, activates turnover of skin cells, and leads to proliferation of skin 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 generates hydrogen and is used for keratin production to improve wrinkles, protect against talmi, and form a keratinous barrier for atopic dermatitis.

Next, fermented grape seed shoots, soybean powder and fermented natto natto bacteria fermented fermented with Benikouji fungus made of red rice honpo, and hydrogen that consists of a protease treatment process A method for producing a resveratrol derivative that is generated and exhibits a keratin producing action will be described.

The resveratrol derivative here is the above-mentioned derivative in which 4 molecules of resveratrol are bound. 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 generates hydrogen, acts directly on skin cell genes, and exhibits keratin production.

This manufacturing method is a step of fermenting fermented liquor of grape seed shoots, soybean powder and natto Honpo natto bacteria, and further fermenting with Benikouji bacteria manufactured by Kurisu Honpo, followed by protease treatment Consists of.

The raw materials are grape seed shoots, soybean powder, Bacillus natto, Bacillus subtilis and protease.

Grape seed shoots are germinated grape seeds. Grape seeds originally contain resveratrol and procyanidins but are bound to dietary fiber and protein.

Since the shoots of grape seeds germinate, the amount of resveratrol increases and it is easy to release, so it is preferable as a source of resveratrol.

Grape seed shoots are obtained by germinating Japanese or Asian grape seeds in clean containers. These seeds are preferably produced with low pesticides or reduced pesticides.

Grape seed shoots are preferably dried and pulverized, and autoclaving prior to fermentation is preferred because the fermentation is carried out smoothly.

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

The soybean powder used as a raw material can be used in soybeans of any origin such as Japanese, Chinese, American, and Russian, but is preferably Japanese because of its reliable traceability and clear producers.

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

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

Furthermore, it is preferable to sterilize the shoots and soybeans of grape seeds after pulverization by autoclaving or the like because they can prevent the propagation of various bacteria.

The natto bacterium produced by Natto Honpo is the scientific name Bacillus subtilis, which is widely used in the production of natto in Japan. Bacteria species from Japan such as Okinawa and Kagoshima, and from Southeast Asia such as China and Taiwan are used. The Bacillus natto used is manufactured by Natto Honpo and exhibits high fermentability.

This Bacillus natto liberates resveratrol in grape seed shoots.

The amount of each of the above-mentioned fermentations is preferably 0.02 to 7 wt. For soybean powder and 0.001 to 0.05 wt. It is preferable to pre-cultivate natto bacteria before being fermented, because the initial time of fermentation is shortened and the fermentation time is shortened.

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 36-42 degreeC, and fermentation is performed for 20 days from 2 days. It is preferable to quantify the target resveratrol derivative by HPLC or TLC and to carry out the fermentation process control while confirming the growth of the cells.

The resulting Bacillus fermentation broth is subsequently fermented by Benikouji fungi manufactured by 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, 0.0001-0.005 weight is preferable with respect to 1 weight of said fermented products. 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 36-43 degreeC, and fermentation is performed for 15 days from 1 day.

Resveratrol tetramer is formed by this fermentation process.

Proteases are hydrolytic enzymes that break down proteins to produce peptides and amino acids, and are also used as food. Amano's protease N is preferred because of its high enzyme activity.

Resveratrol and protein are decomposed by adding a protease to the fermented product and heating it to form a resveratrol derivative.

The amount of protease added is preferably 0.001 to 0.07 weight with respect to 1 weight of the fermented product. The heating temperature is preferably 36 to 43 ° C. The heating time is preferably 1 to 6 hours.

It is preferable that the protease-treated decomposition product is extracted with water-containing ethanol because the product can be efficiently recovered, the protease can be deactivated, and the next step can be easily performed.

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 30 times, more preferably 3 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 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.

Delaware seeds from Yamanashi were soaked in tap water, placed in a clean incubator, and sown in a water-soaked container. This was cultured and germinated at 38 ° C. for 7 days in tap water. The grape seed shoots were collected and dried in a dryer to obtain 1.2 kg of dried grape seed shoots.

The soybeans from Hokkaido were used in a mixer (Cuisinate) to obtain 1.2 kg of soybean pulverized product. The grape seed shoots and soybean grinds 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 12 kg of sterilized tap water was added and stirred.

Separately, 12 g of powdered natto bacteria manufactured by Natto Honpo was used in a small fermentation tank to prepare a sterilized soybean powder and a precultured culture solution.

The pre-cultured Bacillus natto solution is added to the fermented tank containing the dried grape seed shoots and soybeans, and after stirring, warmed in a temperature range of 41-42 ° C. and fermented for 5 days. It was.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration.

10 g of Benikouji fungus made by Kurisu Honpo was added to 1 kg of the fermented product of Bacillus and fermented at 37 ° C. for 5 days.

10 g of Amano Protease N was added to 1 kg of the obtained fermented product and heated at 39 ° C. for 3 hours.

This treated product was heated and ethanol was added to obtain 241 g of the desired resveratrol derivative-containing extract.

1 L of 7% ethanol-containing purified water was added to 200 g of the aforementioned resveratrol derivative-containing extract, and 200 g of Diaion (manufactured by Mitsubishi Chemical) was suspended in a 7% ethanol solution and packed in a column.

3 L of 7% ethanol solution was added thereto for cleaning, and 2 L of 50% 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, 4 molecules of resveratrol were detected from specimen 1. Further structural analysis identified the structure of this resveratrol derivative.

That is, all four molecules of resveratrol were bonded at the 3rd and 5th positions, and no hydroxyl group was observed at this position. On the other hand, the 4-position hydroxyl group was recognized in all 4 molecules. Thereby, a polymer of 4 molecules was confirmed.

Furthermore, as a result of analysis by 400 MHz H-NMR, chemical shift peaks were 1.14, 2.26, 2.56, 3.98, 4.23, 6.08, 6.57, 6.77 and 7. .50 ppm. Since this coincided with the value of the standard product of this resveratrol derivative, it exhibited the structure of this resveratrol derivative.

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

Human skin epithelial cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using 5% fetal calf serum-containing MEM medium (manufactured by Sigma) were seeded in a 35 mm culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas. To this, specimen 1 obtained in Example 1 above and EGF (epidermal growth factor) at a final concentration of 0.1 mg / ml as a control 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 Sample 1 increased the number of epithelial cells in the skin to an average value of 322% compared to the control group. EGF increased by 155%, and specimen 1 was superior.

About the amount of keratins, it increased to 311% by the sample 1 compared with the control group. EGF was 155%, and the increase due to specimen 1 was significant. The cells were not damaged and the safety was confirmed.

The test for confirming the action of generating hydrogen is described below.
(Test Example 3)

100 mg of purified water was added to 1 mg of Sample 1, dissolved, and the hydrogen gas concentration was immediately measured by gas chromatography (Shimadzu Corporation).

As a result, the hydrogen gas concentration of purified water alone was 0.01 ppm, but the purified water to which specimen 1 was added was 5.41 ppm, and generation of hydrogen gas was observed.

The resveratrol derivative obtained in the present invention generates hydrogen and increases keratin, thereby protecting the tissue of cells and protecting the tissue, thereby contributing to the maintenance of national health.

The resveratrol derivative obtained in the present invention has an action to improve skin epithelial cells and keratin, and therefore contributes to the 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)

The resveratrol derivative which generate | occur | produces hydrogen shown by following formula (1), and exhibits keratin production effect | action.

Fermented with fermented grape seed shoots, soybean powder and natto honto natto bacteria, fermented with Benikouji bacterium from red rice honpo, and further generate hydrogen from the process of protease treatment to generate keratin A method for producing a resveratrol derivative exhibiting a production action.