JP6369751B2 - Curcumin derivative exhibiting keratin producing action and method for producing the same - Google Patents

Description

The present invention relates to a curcumin derivative exhibiting 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 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 curcumin derivative having a keratin producing action represented by the following formula (1).

In order to achieve the above object, the invention described in claim 2 relates to a method for producing a curcumin derivative represented by formula (1) according to claim 1 having a keratin producing action.

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

The curcumin derivative according to claim 1 is excellent in keratin production action.

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

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

The curcumin derivative exhibiting a keratin producing action has a structure represented by the following formula (1).

As shown in the formula (1), two molecules of guanine and two molecules of cysteine are bonded to one molecule of curcumin. Cysteine is the L-form.

Curcumin is a kind of polyphenol and has CAS number 458-37-7 and is 1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione.

Its molecular weight is C21H20O6, consisting of 21 carbons, 20 hydrogens and 6 oxygens.

Curcumin changes into dione type and enol type depending on the shape of oxygen, and the action of oxygen exerts a strong antioxidant effect. This curcumin is present in the roots and rhizomes of turmeric.

This curcumin derivative is bonded to one molecule of phenolic hydroxyl group of curcumin through nitrogen and oxygen at the 9th position of guanine. This bond occurs at two locations, and diagonal phenolic hydroxyl groups are bonded.

In addition, two molecules of cysteine are attached to curcumin. This bond is an ester bond between the carboxyl group of cysteine and the phenolic hydroxyl group of curcumin. Again, it is bound to a diagonal phenol group.

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

Further, when a person comes into contact with or drinks an excessive amount of the curcumin derivative, it is decomposed by an enzyme such as esterase in the body and decomposed into curcumin and adenine, which is highly safe.

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

Since this curcumin derivative passes through the cell membrane and further passes through the nuclear membrane and acts on the gene, it is preferable because its action is direct and efficient. In addition, activation that stimulates the active center of keratin-producing enzyme also occurs, and it is preferable because keratin production is increased by a method with a different mechanism, which works on various types of skin.

This curcumin derivative activates and amplifies the gene for keratinase, so that keratinase is induced and keratin production increases. That is, the guanine site of this curcumin derivative binds to the promoter site of the keratinase gene gene, causing gene amplification.

Moreover, the cysteine site of this curcumin derivative is preferable because it adjusts the electrical balance to the enzyme activity center of the keratinase and stimulates the enzyme activity directly.

Furthermore, this curcumin derivative activates the differentiation and proliferation of immature skin cells. The increase in skin cells by this curcumin derivative is due to the activation mechanism at the gene level.

This curcumin derivative is preferred because it activates skin cell turnover and leads to proliferation of skin cells.

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

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, 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 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 against atopic dermatitis.

Next, the curcumin shown by Formula (1) which exhibits the keratin production action which consists of the process which ferments the fermented liquor which added the fermented turmeric rhizome, soybean powder, and natto bacteria with Beniculium fungus, and also protease treatment. A method for producing the derivative will be described.

The curcumin derivative represented by the formula (1) here is the above-mentioned derivative from one molecule of curcumin, two molecules of guanine and two molecules of cysteine. Since this curcumin derivative is decomposed and excreted by an enzyme in vivo, it is highly safe.

This curcumin derivative curcumin 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 subjecting a fermented liquor fermented by adding turmeric rhizome, soybean powder and natto bacteria to fermenting with Benicodorus fungus, followed by protease treatment.

The raw materials are turmeric rhizome, soybean powder, Bacillus natto, Beniculum and protease.

Turmeric is a perennial plant belonging to the genus Turmeric belonging to the family Ginger, and is also referred to as autumn turmeric, chrysanthemum or turmeric, and has the scientific name Curcuma longa. Turmeric rhizome is used for food and medicine, and is highly safe.

Turmeric rhizome is preferred as a raw material for this derivative because it contains abundant curcumin.

Turmeric rhizomes are good from Japan, from Asia such as China, and from America. Of these, those produced with low pesticides or reduced pesticides are preferred.

Turmeric rhizomes are preferably dried and pulverized, and sterilization by autoclave prior to 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 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.

Further, turmeric rhizomes and soybeans are preferably sterilized after pulverization by an autoclave or the like because they can prevent the propagation of various bacteria.

The Bacillus natto used 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.

This Bacillus natto is fermented with curcumin consisting of turmeric rhizome and soybean and base of nucleic acid at the same time, and curcumin and base are combined.

The amount of each of the fermentations is preferably 0.02 to 5 weights for soybean powder and 0.001 to 0.05 weights for Bacillus natto with respect to 1 weight of dry powder of turmeric rhizome. 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 35-44 degreeC, and fermentation is performed for 2 to 40 days. It is preferable to carry out the process control of the fermentation by quantifying the target curcumin derivative by HPLC or TLC and confirming the growth of the cells.

The resulting Bacillus fermentation broth is subsequently fermented by B. niger. The curcumin derivative is separated by the fermentation by the fungus, and absorption is also promoted.

The fungus 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, 0.001-0.006 weight is preferable with respect to 1 weight of said fermented products. It is preferable to pre-cultivate B. subtilis before fermentation 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 36-43 degreeC, and fermentation is performed for 1 day to 34 days.

By this fermentation process, a nucleic acid and a conjugate such as an ester bond are formed.

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.

When the protease is added to the fermented product and heated, the curcumin derivative and protein are decomposed into a low molecular weight curcumin 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 35 to 46 ° C. The heating time is preferably 1 hour to 7 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 target curcumin 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 curcumin 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 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 a fraction containing the curcumin derivative and remove the solvent by drying or vacuum drying to obtain the target curcumin 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 curcumin 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 curcumin 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.

The rhizome of autumn turmeric grown in Okinawa with reduced pesticides was used. After collecting the rhizome, washed with tap water, dried in the sun, and pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.5 kg of pulverized dried powder of turmeric rhizome It was.

The soybeans from Hokkaido were used in a mixer (Cuisinate) to obtain 1.1 kg of soybean pulverized material. The turmeric rhizome and soybean ground product were subjected to an autoclave and sterilized at 121 ° C. for 20 minutes.

These were put into a clean fermentation tank (sterilized round 40-liter tank for fermentation), and 10 kg of sterilized tap water was added and stirred.

Separately, 10 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 was added to the fermentation tank containing the dried turmeric rhizome powder and soybeans, and after stirring, heated in a temperature range of 41 to 42 ° C. for fermentation.

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 38 ° C. for 5 days.

10 g of Amano Protease N was added to 1 kg of the obtained fermented product and heated at 40 ° C. for 3 hours.

This treated product was heated, and ethanol was added to obtain 520 g of the target curcumin derivative-containing extract.

1 L of 6% ethanol-containing purified water was added to 400 g of the curcumin derivative-containing extract described above, and 400 g of Diaion (manufactured by Mitsubishi Chemical) was suspended in a 5% ethanol solution and packed in a column.

To this, 2 L of 5% ethanol solution was added for cleaning, and 2 L of 50% ethanol solution was added to elute the desired curcumin derivative and purified. The purified curcumin derivative was distilled under reduced pressure to remove the ethanol portion to obtain an aqueous solution. This was designated as specimen 1 of the curcumin derivative.

Below, the test method and result regarding the structural analysis of a curcumin derivative 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, curcumin and base adenine were detected from specimen 1. In addition, 1 molecule of curcumin, 2 molecules of guanine, and 2 molecules of cysteine were confirmed, and the respective bindings were confirmed. Further structural analysis identified the structure of this curcumin derivative.

That is, the 9th position of guanine was bonded to one molecule of the phenolic hydroxyl group of curcumin, and the bond was found at two positions, and the positions were diagonal. Furthermore, an ester bond between one molecule of the phenolic hydroxyl group of curcumin and cysteine was confirmed. The binding was also diagonal, and guanine and cysteine were in close proximity.

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, the cell suspension was adjusted, 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 epithelial cells in the skin to an average value of 390% compared to the control group. EGF increased by 140%, and specimen 1 was superior.

About the amount of keratin, it increased to 410% by the sample 1 compared with the control group. EGF was 150%, and the increase effect of specimen 1 was remarkable. The cells were not damaged and the safety was confirmed.

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

Since the curcumin derivative obtained in the present invention has the action of improving skin epithelial cells and keratin, it contributes to the improvement of those suffering from inflammation, atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetics industry.

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

Claims (2)

The curcumin derivative which exhibits the keratin production effect | action shown by following formula (1).
Rhizome turmeric, a fermentation solution obtained by fermentation by adding soybean powder and natto is further fermented with Monascus fungi, further represented by the formula of claim 1 exhibiting keratin production action comprises the step of protease treatment (1) A method for producing a curcumin derivative.