JP6741965B2 - Dehydroretinol derivative exhibiting keratin increasing action - Google Patents

Description

The present invention relates to a dehydroretinol derivative exhibiting a keratin increasing action.

Keratin is a protein that forms an intracellular skeleton and a hard protein that forms tissues such as skin and blood vessels, and plays an important role in maintaining bone and skin tissues. Keratin is biosynthesized in the body by stimulating epithelial cells and regenerating cells.

The keratin contained in the skin is involved in the protection of the keratin and the arrangement of epithelial cells, and cosmetically affects the arrangement and arrangement of the cuticles and sulci of the skin, and thus has a relationship with wrinkles and sagging of the skin. strong. In addition, maintenance and increase of keratin are essential for maintenance of hair.

Furthermore, keratin also plays an important role as a tissue constituent protein in non-keratinizing epithelial cells, and the sheet-like structure of epithelial tissue maintains its mechanical strength by keratin fibers. A highly safe natural product that leads to an increase in keratin is desired.

For example, there is a use of a heterocyclic quaternary polyammonium polymer as a protective agent for keratin fibers and a cosmetic composition (see, for example, Patent Document 1). However, its range of use is limited, and no strong action such as a direct action on skin cells has been shown.

Patent No. 3823025

The keratin-increasing effect of existing plant extracts and chemical substances is mild, and there is a problem that their industrial use is limited.

In addition, chemically synthesized substances have safety problems and their use is limited.

Therefore, a natural product having weak side effects and exhibiting an excellent keratin increasing action is desired.

In order to achieve the above object, the invention according to claim 1 relates to a dehydroretinol derivative having the keratin-increasing action represented by the following formula (1).

Since the present invention is configured as described above, it has the following effects.

According to the dehydroretinol derivative of the first aspect, a natural product exhibiting a keratin-increasing action with few side effects can be obtained.

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

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

The dehydroretinol derivatives shown here act directly on human skin cells and hair cells to increase keratin production. Its action involves the proliferation of skin epithelial cells and the induction of keratin-producing enzymes.

This dehydroretinol derivative is composed of hydroxyretinol and L-cysteine. The molecular formula is C26H38O3N2S2, which is composed of 26 carbons, 38 hydrogens, 3 oxygens, 2 nitrogens and 2 sulfurs. That is, two molecules of L-cysteine are bound to one molecule of hydroxyretinol.

This dehydroretinol derivative can be synthesized organically from retinol and cysteine, and can be used as a standard product for structural analysis. In addition, Diaion (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas), Sephadex LH-20 (manufactured by Amersham Pharmacia), ion exchange carrier IRA-410 (Rohm and). Purified with a reverse phase carrier DM1020T (manufactured by Fuji Silysia) and a purified product having a purity of 95% or more can be obtained.

This structure can be analyzed by analyzing 1H-NMR and 13C-NMR in CD3OD with a nuclear magnetic resonance apparatus (for example, NMR, manufactured by Bruker). By 1H-NMR analysis at 600 MHz, 1.469, 1.514, 1.661, 1.887, 1.955, 2.079, 2.102, 2.124, 2.192, 2.215, 2.15. 305, 2.407, 2.565, 2.599, 2.644, 2.667, 2.712, 2.893, 3.040, 3.254, 3.367, 3.763, 4.023, Peaks are observed at 4.068, 5.119, 6.147, 6.283, 7.006 and 7.379 ppm.

Furthermore, by analysis of 13 C-NMR in CD3OD, 23.0, 26.7, 27.1, 27.3, 28.7, 30.0, 34.1, 39.9, 41.6, 46.7. , 49.4, 51.6, 55.3, 58.8, 71.8, 72.6, 78.8, 129.6, 135.1, 141.0, 142.5 and 176.2 ppm. Is recognized.

Dehydroretinol is a kind of carotenoid, which is a natural product that has an antioxidant action and an action on the optic nerve, and also acts on the growth and maintenance of cells in the skin. In the natural world, dehydroretinol is produced from retinol by an enzyme, decomposed in vivo, and excreted, so that it is highly safe. In addition, it has no accumulation in the body or the environment and is highly safe to the environment.

In the dehydroretinol derivative shown here, the hydroxyl group of dehydroretinol and the carboxyl group of L-cysteine are ester-bonded. Moreover, since it is easily ionized, it is excellent in absorption into the skin and absorption from the intestinal tract. Since this dehydroretinol derivative contains L-cysteine, which is a constituent of keratin, it is also useful as a raw material for keratin.

Furthermore, the dehydroretinol derivative activates the epidermal growth factor receptor of skin epithelial cells. The activation method is to improve the sensitivity, which is so-called hypersensitivity, which acts on the active center of the enzyme and enhances the sensitivity as a catalyst.

An excessive amount of this dehydroretinol derivative is decomposed by nonspecific esterase existing in the liver and kidney, and decomposed into dehydroretinol and L-cysteine.

Since both dehydroretinol and L-cysteine exist in the natural world and their safety has been confirmed, the safety of this dehydroretinol derivative is high.

In addition, this dehydroretinol derivative is neither accumulated in fat nor concentrated.

Furthermore, this dehydroretinol derivative has an effect of improving blood flow, a detox effect, an excretion effect and a metabolic activation effect, in addition to proliferating epithelial cells and hair matrix cells of the skin and increasing keratin production.

As the method for producing the dehydroretinol derivative, it is possible to obtain it by fermenting caviar, vegetables, and algae, in addition to the method of organically synthesizing.

Of these, caviar contains abundant retinol and further contains epidermal growth factor that promotes the growth of skin cells and hair matrix cells, which is preferable. Caviar is an egg of sturgeon whose scientific name is Acipenser medirostris. It can be used with salted caviar as it does not affect retinol even when salted.

This fermentation process consists of adding natto bacteria and red yeast mold to ferment.

Next, it is fermented by Bacillus natto and Aspergillus niger. That is, caviar, rice bran, Bacillus natto and Aspergillus niger are added to a clean fermentation tank and fermented under aerobic conditions.

The rice bran paste used as a raw material can be rice bran rice produced from brown rice in any of the production areas such as Japan, China, USA, Russia, etc. However, Japan is preferable because the traceability is reliable and the producer is clear. Among these, brown rice-derived rice bran grown organically or without pesticides is more preferable because it does not contain harmful pesticides or metals.

When using Komenuka, Nara Kikai Seisakusho Co., Ltd.'s free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko as a vacuum dryer, small size manufactured by Matsui Co., Ltd. It is pulverized by a pulverizer such as a reduced pressure heat transfer dryer DPTH-40, AQM Kyushu Technos Co., Ltd. clean dry VD-7, VD-20, and Nakayama Technical Research Institute DM-6. This pulverization is preferable because the fermentation process can easily proceed efficiently.

The natto bacterium used has a scientific name of Bacillus subtilis and is a useful edible bacterium that is widely used in Japan for the production of natto and food processing and has abundant food experience. Bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia from China and Taiwan are used. Of these, natto bacterium produced by Natto Honpo is preferable because it exhibits high fermentability.

The Monascus fungus to be used has a scientific name of Monascaceae and is a useful edible fungus with abundant food experience. Bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia from China and Taiwan are used. Of these, the red mold fungus manufactured by Red Koji Honpo is preferable because it exhibits high fermentability.

The addition amount of each of the fermentations is preferably 0.4 to 5 weights for rice bran, 0.002 to 0.04 weights for Bacillus natto, and 0.001 to 0.05 weights for Aspergillus niger relative to 1 weight of caviar. .. It is preferable to pre-culture the Bacillus natto and Aspergillus niger before they are fermented because the initial time of fermentation is shortened and the fermentation time is shortened.

The fermentation is preferably carried out in a clean culture tank and the materials are preferably mixed with sterilized tap water.

Further, this fermentation is heated to 35 to 48° C., and the fermentation is carried out for 4 to 14 days.

After fermentation, the natto bacteria and red yeast mold are killed by heating at about 90°C, and the fermentation is stopped. This process of fermentation releases dehydroretinol. Hydroxyproline is then released from the rice bran.

It is preferable that the fermented product produced by the above fermentation is extracted with hydrous ethanol because the product can be efficiently recovered and the next step can be easily carried out. In addition, it is preferable to subject the obtained fermented product to ultrasonic disruption because the product is easily separated. Further, concentration by freeze-drying or the like is preferable because the following steps can be carried out in a short time.

It is preferable to separate and purify the fermented product from the viewpoint that the intake can be reduced as a highly pure substance. As a method for this purification, it is preferable to utilize a purification operation of a resin for separation or the like.

For example, it is preferable that the carrier is separated and separated by a carrier for separation or a resin. As the carrier or resin for separation, porous polysaccharides, silicon oxide compounds, polyacrylamides, polystyrenes, polypropylenes, styrene-vinylbenzene copolymers, etc., the surfaces of which are coated as described later, are used. Those having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the more accurate the separation is performed, but the separation time is long.

For example, a reverse phase carrier or a resin whose surface is coated with a hydrophobic compound is used for separating a highly hydrophobic substance. Those coated with cationic substances are suitable for separating anionically charged substances. Also, those coated with an anionic substance are suitable for separating cationically charged substances. When coated with a specific antibody, it is used as an affinity carrier or resin for separating only a specific substance.

Affinity carriers or resins are used for the specific preparation of antigens using the antigen-antibody reaction. A distributable carrier or resin is used for isolation of substances such as silica gel (manufactured by Merck & Co., Inc.) when there is a difference in the distribution coefficient between the substance and the separating solvent.

Among these, an adsorbent carrier or resin, a distributable carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable because they can reduce the production cost. Furthermore, the reverse phase carrier or resin and the distributable carrier or resin are more preferable because the difference in partition coefficient with respect to the separating solvent is large.

When an organic solvent is used as the separating solvent, a carrier or resin having resistance to the organic solvent is used. In addition, carriers or resins used for pharmaceutical production or food production are preferable.

From these points, Diaion (HP-20 type or HP21 type, manufactured by Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (produced by Rohm and Haas) as an adsorptive carrier, and Sephadex LH as a carrier for molecular sieves. -20 (manufactured by Amersham Pharmacia), silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier are more preferable.

Among these, Diaion HP-20 type, Sephadex LH-20 and DM1020T are more preferable.

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. From the viewpoint of separation efficiency, the amount thereof is preferably 1 to 35 times the weight of the extract, and more preferably 4 to 25 times. 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 separating 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, the separating solvent is preferably a lower alcohol. When silica gel is used, the separating solvent is preferably chloroform, methanol, acetic acid or a mixture thereof.

When using Diaion HP-20 type and DM1020T, the separating solvent is preferably a lower alcohol such as methanol or ethanol, or a mixture of lower alcohol and water.

Further, it is preferable to collect the fraction containing the activity and remove the solvent by drying or vacuum drying to obtain a powder or a concentrated solution because the influence of the solvent can be excluded.

Further, it is preferable to carry out the final extraction with an edible oil or a fat or oil used for cosmetics, since the obtained active portion is stably maintained in the oil. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil, jojoba oil is preferable.

It is used as a pharmaceutical as a parenteral agent such as an injection or an oral agent or a coating agent, and is used as a quasi-drug compounded in tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. It

Examples of oral agents include tablets, capsules, powders, syrups, drinks and the like. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, an excipient, a swelling agent, a lubricant, a sweetener, a flavoring agent and the like. The tablets can also be coated with shellac or sugar.

Further, in the case of the above-mentioned capsule, the above-mentioned material may further contain a material such as oil and fat. In the case of the above-mentioned syrups and drinks, sweeteners, preservatives, coloring and flavoring agents can be added.

Examples of parenteral agents include injections as well as external preparations such as ointments, creams and solutions. As a base material for the external preparation, petrolatum, paraffin, oils and fats, lanolin, macro gold and the like are used, and an ointment, a cream or the like can be prepared by a usual method.

Injections include liquid preparations and other freeze-dried preparations. It is used by aseptically dissolving it in distilled water for injection, physiological saline, etc. before use.

It is used as a food formulation for beauty foods for beauty, foods for beauty, and nutritional tonics for maintaining liver cells. Moreover, it is preferable to use it as a nutritionally functional food or a food for specified health care as a food with health claims.

When the obtained food preparation is used for pets such as dogs and cats and livestock animals, it is used as feed or supplement for the purpose of maintaining skin health.

It can be used as a cosmetic 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, face wash, beauty essence, lotion, etc., and it is a cosmetic that promotes keratin production. The cosmetic may be in any form and can be used in the form of solution, cream, paste, gel, gel, solid or powder.

It can also be used as a plant activator for the purpose of activating plants energetically by a mechanism of increasing keratin that forms the skeleton of plants. It can also be used in beans, grains, rice, root vegetables and flowers, increasing keratin in plants to improve yield and quality, and increase plant growth and longevity. It can also be used to hold cut flowers.

Hereinafter, the said embodiment is concretely demonstrated using an Example and a test example. It should be noted that these are merely examples, and the conditions can be changed within the range of common sense according to the difference in materials, raw materials, and samples.

We purchased and used caviar collected from cultured sturgeon from Okayama prefecture. 450 g of this was stirred with a mixer.

The resulting caviar suspension was placed in a clean fermentation tank (Owaki Engineer, 150 kg capacity).

1.1 kg of rice bran obtained from brown rice grown in Akita prefecture with reduced pesticides was purchased and suspended in 9 L of tap water. This rice bran suspension was placed in an autoclave (Tomy Precision, SR-240) and sterilized at 121°C.

After sterilization, the cooled rice bran liquid was transferred to a fermentation tank, 8 g of Natto bacteria and 10 g of Aspergillus niger manufactured by Natto Honpo were added, and fermented at 37° C. for 4 days.

The fermented liquor was placed in a heating tank at 91°C and heated for 14 minutes to sterilize it. This was cooled and then filtered to obtain 8.1 L of a filtrate. This was designated as a dehydroretinol derivative-containing extract. Stored at 4°C until use.

3 L of 5% ethanol-containing purified water was added to 4 L of the above-mentioned dehydroretinol derivative-containing extract, and 200 g of Diaion HP-20 type (manufactured by Mitsubishi Chemical) was suspended in a 4% ethanol solution and applied to a column packed.

To this, 3 L of 4% ethanol solution was added for cleaning, and 2 L of 40% ethanol solution was added to elute the target dehydroretinol derivative. The ethanol portion of the purified dehydroretinol derivative was distilled off under reduced pressure to obtain an aqueous solution. Further, this purification step was repeated 3 times to obtain a dehydroretinol derivative specimen 1 having a purity of 97.3%. This sample 1 exhibited a single peak by HPLC analysis.

The identification test of the dehydroretinol derivative will be described below.
(Test Example 1)

The sample 1 which is the dehydroretinol derivative of Example 1 obtained as described above was dissolved in purified ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation), and further, a nuclear magnetic resonance apparatus (NMR , Manufactured by Bruker).

As a result, by 1H-NMR analysis at 600 MHz in CD3OD, 1.469, 1.514, 1.661, 1.887, 1.955, 2.079, 2.102, 2.124, 2.192, 2.215, 2.305, 2.407, 2.565, 2.599, 2.644, 2.667, 2.712, 2.893, 3.040, 3.254, 3.367, 3. Peaks were observed at 763, 4.023, 4.068, 5.119, 6.147, 6.283, 7.006 and 7.379 ppm.

Furthermore, by analysis of 13 C-NMR in CD3OD, 23.0, 26.7, 27.1, 27.3, 28.7, 30.0, 34.1, 39.9, 41.6, 46.7. , 49.4, 51.6, 55.3, 58.8, 71.8, 72.6, 78.8, 129.6, 135.1, 141.0, 142.5 and 176.2 ppm. Was recognized.

The chart of 13 C-NMR analysis results is shown below. (The horizontal axis unit is ppm, and the vertical axis unit is peak intensity.)

That is, the sample of Example 1 showed the same analysis result as the standard product synthesized and purified organically. It was identified as the desired dehydroretinol derivative.

The confirmation test using human skin epithelial cells will be described below.
(Test example 2)

Human skin epithelial cells purchased from Kurabo Co., Ltd. were used. As a culture medium, MEM culture medium (manufactured by Sigma) containing 5% fetal bovine serum was used. 1000 cells were seeded on a 35 mm culture dish and cultured at 37° C. in 5% carbon dioxide gas. To this, the specimen 1 obtained in Example 1 and the skin epidermal growth factor (EGF) as a control were added at a final concentration of 0.1 mg/ml. This was cultured for 48 hours. EGF is used as a physiological substance that proliferates epithelial cells of the skin and produces keratin. After detaching the cells, the number of cells was counted. In addition, the average value was calculated using five petri dishes.

As a result, the addition of 0.1 mg/ml of Sample 1 increased the number of epithelial cells in the skin to 255% as an average value compared with the solvent control group. In addition, as a result of quantifying the amount of keratin with an ELISA kit (manufactured by Wako Pure Chemical Industries, Ltd.), the average value was 344% compared with the solvent control group.

Further, the number of cells in the control EGF was 181% compared to the control group. In addition, the amount of keratin produced was 210%, and Sample 1 was superior to the increase in keratin amount.

On the other hand, in the skin irritation experiment using EpiSkin (manufactured by SkinEthic) which is artificial skin as part of the safety test, irritation was not recognized by addition of the sample 1, and safety was confirmed. Note that this method has been established as a method that does not use animals as a skin irritation test evaluation method using cells.

The confirmation test as a plant activator using perilla seeds and soybean seeds will be described below.
(Test Example 3)

Perilla seeds and soybean seeds from Nagano prefecture were purchased and used. The perilla seeds and the soybean seeds were placed on a gauze-lined vat in which a 1000-fold diluted aqueous solution of Sample 1 was sufficiently dipped. This was placed in an incubator at 30° C. and cultured for 7 days, and the spread of buds was measured. The average value of 10 seeds was determined. Only tap water was used as a solvent control. At the same time, as a control, the culture was immersed in a 1000 times solution of HB-101 (manufactured by Flora Co., Ltd.).

As a result, the buds of perilla seeds were 146% when immersed in Specimen 1 and 131% when immersed in HB-101 when the solvent control was 100%. Soybean seeds spread 150% when immersed in Specimen 1 compared to the solvent control. On the other hand, when immersed in HB-101, it was 126% of the solvent control. From these results, it was concluded that Specimen 1 exhibits a superior plant activating action than HB-101.

Since the dehydroretinol derivative obtained by the present invention exhibits a keratin-producing effect and has few side effects, it can improve the QOL of the people, increase the healthy working population, and reduce medical costs.

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

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

The dehydroretinol derivative obtained in the present invention is used as a plant activator to increase plant extension and growth, fruit set and yield, and is also used for desert greening measures and agricultural fields.

Claims (1)

A dehydroretinol derivative having a keratin-producing action represented by the following formula (1).